Novel usp7 inhibitors for treating multiple myeloma

ABSTRACT

The present disclosure relates to inhibitors of USP7 useful in the treatment of cancers, and other USP7 mediated diseases, having the Formula: 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and n are described herein.

RELATED APPLICATIONS

This application is the § 371 National Stage of PCT/US2018/052797, filedSep. 26, 2018; which claims the benefit of U.S. Provisional PatentApplication No. 62/563,375, filed on Sep. 26, 2017. The contents of eachof these applications are hereby incorporated by reference in theirentirety.

STATEMENT OF RIGHTS

This invention was made with government support under Grant RO1 CA211681awarded by the National Institutes of Health. The U.S. government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 24, 2020, isnamed DFS-18425_SL.txt and is 3,938 bytes in size.

BACKGROUND

Deubiquitinating enzymes (DUBs) have garnered significant attention asdrug targets in the last 5-10 years. DUB inhibitors effectively promotedegradation of oncogenic proteins, especially proteins that arechallenging to directly target because they are stabilized by DUB familymembers. Highly-optimized and well-characterized DUB inhibitors havethus become highly sought after tools. Most reported DUB inhibitors,however, are polypharmacological agents possessing weak (micromolar)potency toward their primary target, thereby limiting their utility intarget validation and mechanism studies. Due to a lack of highresolution DUB⋅small molecule ligand complex structures, nostructure-guided optimization efforts have been reported for a mammalianDUB.

The DUB enzyme USP7 has been shown to be involved in regulation of amyriad of cellular processes, including epigenetics, cell cycle, DNArepair, immunity, viral infection and tumorigenesis. USP7, also known asherpes virus-associated ubiquitin specific protease (HAUSP), was firstdiscovered as a protein that plays a role in viral lytic growth.(Everett et al., Novel ubiquitin-specific protease is dynamicallyassociated with the PML nuclear domain and binds to a herpesvirusregulatory protein. EMBO J, 16, 1997, 566-77.) Interest in the enzymeintensified when USP7 was implicated in regulating degradation of thetumor suppressor p53, by stabilizing the major E3 ligase for p53, MDM2.(Li et al., Deubiquitination of p53 by HAUSP is an important pathway forp53 stabilization. Nature, 416, 2002, 648-53; Cummins et al., Tumoursuppression: disruption of HAUSP gene stabilizes p53. Nature, 428, 2004,1 p following 486; Li et al., A dynamic role of HAUSP in the p53-Mdm2pathway. Mol Cell, 13, 2004, 879-86).

Consistent with its regulation of diverse substrates and biologicalprocesses USP7 has emerged as a drug target in a wide range ofmalignancies including multiple myeloma, breast cancer, neuroblastoma,glioma, and ovarian cancer. (Chauhan et al., A small molecule inhibitorof ubiquitin-specific protease-7 induces apoptosis in multiple myelomacells and overcomes bortezomib resistance. Cancer Cell, 22, 2012,345-58; Wang et al., J Clin Invest, 126, 2016, 2205-20; Tavana et al.,Nat Med, 22, 2016, 1180-1186; Cheng et al., Expression of HAUSP ingliomas correlates with disease progression and survival of patients.Oncol Rep, 29, 2013, 1730-6; Zhang et al., Expression of USP7 and MARCH7Is Correlated with Poor Prognosis in Epithelial Ovarian Cancer. Tohoku JExp Med, 239, 2016, 165-75) However, known USP7 inhibitors have beenshown to exhibit modest potency against USP7 and poor selectivity overother DUBs. In addition to modest potency and selectivity, reporteddrawbacks of known USP7 inhibitor compounds include poor solubility andgeneral toxicity. (Chen et al., Synthesis and biological evaluation ofthiazole derivatives as novel USP7 inhibitors. Bioorg Med Chem Lett, 27,2017, 845-849). Therefore, there is a need for the development of morepotent, selective, soluble USP7 inhibitors with reduced toxicity.

SUMMARY

Disclosed herein are compounds of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

-   R₁ is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NO₂,    —NH₂, CN, —NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈, wherein each    alkyl is independently optionally substituted with one or more R₉;-   R₂ is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NH₂,    —NO₂, CN, —NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈, wherein each    alkyl is independently optionally substituted with one or more R₉;-   R₃ is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NH₂,    —NO₂, CN, —NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈; wherein each    alkyl is independently optionally substituted with one or more R₉;-   R₄ is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NO₂,    —NH₂, CN, —NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈; wherein each    alkyl is independently optionally substituted with one or more R₉;    wherein R₁, R₂, R₃ and R₄ are not simultaneously H;-   R₅ is H, halogen, —CN, —OR₇, or —NR₇R₈;-   R₆ is alkyl, —C(═O)R₁₀, —C(═S)R₁₀, —C(O)NR₇R₈, cycloalkyl,    heterocycloalkyl, aryl, or heteroaryl; wherein the cycloalkyl,    heterocycloalkyl, aryl, heteroaryl are each independently optionally    substituted with one or more R₁₁; and wherein the alkyl is    substituted with one or more R₁₂;-   each R₇ and R₈ is independently H, alkenyl, or alkyl;-   each R₉ is independently at each occurrence —NR₇R₈, alkoxy,    —(OCH₂CH₂)_(m)alkyl, cycloalkyl, heterocycloalkyl, aryl, or    heteroaryl, wherein the alkyl and alkoxy are each independently    optionally substituted with one or more substituents selected from    alkoxy, haloalkoxy, halogen, and —OH; and wherein the cycloalkyl,    heterocycloalkyl, aryl and heteroaryl are each independently    optionally substituted with one or more substituents selected from    alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —N₃, and —OH;-   R₁₀ is alkyl, alkenyl, alkynyl, —NR₇R₈, cycloalkyl,    heterocycloalkyl, aryl, amino, heteroalkyl, alkylamino, aminoalkyl    or heteroaryl, wherein the alkyl, alkenyl, and alkynyl are each    independently optionally substituted with one or more R₁₃; and    wherein the cycloalkyl, heterocycloalkyl, aryl and heteroaryl are    each independently optionally substituted with one or more R₁₂;-   each R₁₁ is independently at each occurrence alkyl, haloalkyl,    alkoxy, haloalkoxy, halogen, —NO₂, or —OH;-   each R₁₂ is independently at each occurrence aryl or heteroaryl,    wherein the aryl and heteroaryl are each independently optionally    substituted with one or more substituents selected from alkyl,    haloalkyl, alkoxy, haloalkoxy, halogen, and —OH;-   each R₁₃ is independently at each occurrence —OH, alkoxy,    heteroalkyl, aryloxy, —NH₂, arylalkyl, cycloalkyl, heterocycloalkyl,    aryl, heteroaryl, —O-aryl, —O-heteroaryl, —NR₇aryl, —NR₇heteroaryl,    or —NR₇C(═O)R₁₄, wherein the cycloalkyl, heterocycloalkyl, aryl,    heteroalkyl, and heteroaryl are each independently optionally    substituted with one or more substituents selected from alkyl,    haloalkyl, alkoxy, haloalkoxy, halogen, —NO₂, and —OH;-   R₁₄ is alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, cycloalkyl,    aryl, heterocyclyl, or heteroaryl, wherein the aryl and heteroaryl    are each independently optionally substituted with one or more R₁₅;    and wherein the alkyl, alkenyl, and alkynyl are each independently    optionally substituted with one or more substituents selected from    halogen and —OH;-   each R₁₅ is independently at each occurrence halogen, alkyl, CN,    —C(═O)alkyl, or —C(═O)alkenyl, wherein the alkyl and alkenyl is each    independently substituted with one or more substituents selected    from halogen and —OH;-   m is 1, 2, or 3; and-   n is 0 or 1;-   provided that:-   (i) if R₂ is —NO₂, —NHC(O)Me or —NH₂, and R₁, R₂, and R₄ are each H;    or R₁ is Me and R₂, R₃, and R₄ are each H; then R₆ is not —C(O)R₁₀    where R₁₀ is —(CH₂)—(CHMe)-phenyl;-   (ii) when R₂ is Cl, R₁, R₃ and R₄ are each H, R₆ is —C(═O)R₁₀, and    R₁₀ is (C₂-C₃)alkyl substituted with one R₁₃; then R₁₃ is not    unsubstituted cyclopentyl, unsubstituted phenyl or unsubstituted    2-thiophenyl; and-   (iii) when R₂ is Cl, and R₁, R₃ and R₄ are each H; then R₆ is    —C(═O)R₁₀, R₁₀ is not 1-ethylpropyl.

In certain embodiments, the present invention provides a pharmaceuticalcomposition suitable for use in a subject in the treatment or preventionof a disorder associated with modulation of USP7 comprising an effectiveamount of any of the compounds described herein (e.g., a compound of theinvention, such as a compound of formula (I)), and one or morepharmaceutically acceptable excipients. In certain embodiments, thepharmaceutical preparations may be for use in treating or preventing acondition or disease as described herein.

Disclosed herein are methods of inhibiting USP7, comprisingadministering to a subject a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof.

Disclosed herein are methods of treating diseases and conditions thatbenefit from the modulation of USP7, comprising administering to asubject a therapeutically effective amount of a compound of Formula (I),or a pharmaceutically acceptable salt thereof. In some embodiments, thediseases and conditions benefit from the inhibition of USP7. Thesediseases and conditions include, but are not limited to, cancer andmetastasis, neurodegenerative diseases, immunological disorders,diabetes, bone and joint diseases, osteoporosis, arthritis inflammatorydisorders, cardiovascular diseases, ischemic diseases, viral infectionsand diseases, viral infectivity and/or latency, and bacterial infectionsand diseases.

Disclosed herein are methods of treating cancer, comprisingadministering to a subject a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof.In some embodiments, the cancer is multiple myeloma.

Disclosed herein is a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, for use in the manufacture of a medicament fortreating a disease or condition associated with inhibiting USP7.

Disclosed herein is the use of a compound of Formula (I), or apharmaceutically acceptable salt thereof, in the treatment of a diseaseor condition associated with inhibiting USP7.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: Known compound P22077 and its close analog P5091.

FIG. 1B: Known USP7 inhibitors.

FIG. 2A: Structure-guided optimization of compound A led to compounds 10and 11. The enantiomer of compound 10, compound 11, is 80-fold lessactive.

FIG. 2B: Dose-dependent inhibition of the USP7 catalytic domain (aminoacids 208-560) and full-length USP7 (amino acids 1-1102) by compound 10and compound 11 using Ub-AMC as substrate.

FIG. 2C: Assessment of compound 10 binding to USP7 using isothermalcalorimetry.

FIG. 2D: Inhibitory activity of compound 10 across a panel of 41purified DUBs using ubiquitin-rhodamine (Ub-Rho) as substrate.

FIG. 3A: Characterization of compound 10 binding to USP7 using ribbondiagram of USP7 with compound 10.

FIG. 3B: Stereoview of USP7 (light blue) bound to compound 10 (yellow).Hydrogen bonds are indicated by dashed lines.

FIG. 3C: Molecular surface representation of the USP7⋅compound 10co-structure. Highlighted regions indicate regions of altered HDX in thepresence of compound 10. Darker areas correspond to significant changeswhereas lighter areas correspond to regions with subtle changes.

FIG. 4A: Analysis of USP7 mutant proteins. Detailed ligand interactiondiagram of compound 10 with USP7. Residues for which >80% of other USPscontain an amino acid belonging to the same class are boxed red.

FIG. 4B: Summary of activity against Ub-AMC and inhibition by compound Afor USP7 mutant catalytic domain proteins.

FIG. 4C: Dose-response inhibition of full length USP7Q351 (amino acids1-1102) by compound A.

FIG. 4D: Dose-response inhibition of full length USP7Q351 (amino acids1-1102) by compound 10.

FIG. 5A: USP7 inhibitory activity and mouse liver microsome (MLM)stability of disclosed compounds.

FIG. 5B: Structures, USP7 inhibitory activity and mouse liver microsome(MLM) stability of disclosed compounds.

FIG. 5C: Analysis of the ability of compounds 10 and 11 to bind nativeUSP7 across multiple doses in HEK293T lysates using competitive activitybased protein profiling.

FIG. 6A: Compound 10 promotes loss HDM2 and accumulation of p53 and p21.Analysis of HDM2, p53 and p21 protein levels in MCF7 cells treated withcompounds 10 or 11 at the indicated concentration for 16 hours.

FIG. 6B: Analysis of HDM2, p53 and p21 protein levels in MCF7 cellsfollowing 16 hours of treatment with compounds 10 or 11 at the indicatedconcentration with addition of cycloheximide for the last 2 hours.

FIG. 6C: Analysis of HDM2, p53 and p21 protein levels in MM.1S cellstreated with compounds 10 or 11 at the indicated concentration for 6hours.

FIG. 6D: Analysis of HDM2, p53 and p21 protein levels in MM.1S cellsfollowing 6 hours of treatment with compounds 10 or 11 at the indicatedconcentration with addition of cycloheximide for the last 2 hours.

DETAILED DESCRIPTION

Disclosed herein are compounds of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

-   R₁ is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NO₂,    —NH₂, CN, —NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈, wherein each    alkyl is independently optionally substituted with one or more R₉;-   R₂ is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NH₂,    —NO₂, CN, —NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈, wherein each    alkyl is independently optionally substituted with one or more R₉;-   R₃ is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NH₂,    —NO₂, CN, —NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈; wherein each    alkyl is independently optionally substituted with one or more R₉;-   R₄ is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NO₂,    —NH₂, CN, —NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈; wherein each    alkyl is independently optionally substituted with one or more R₉;    wherein R₁, R₂, R₃ and R₄ are not simultaneously H;-   R₅ is H, halogen, —CN, —OR₇, or —NR₇R₈;-   R₆ is alkyl, —C(═O)R₁₀, —C(═S)R₁₀, —C(O)NR₇R₈, cycloalkyl,    heterocycloalkyl, aryl, or heteroaryl; wherein the cycloalkyl,    heterocycloalkyl, aryl, heteroaryl are each independently optionally    substituted with one or more R₁₁; and wherein the alkyl is    substituted with one or more R₁₂;-   each R₇ and R₈ is independently H, alkenyl, or alkyl;-   each R₉ is independently at each occurrence —NR₇R₈, alkoxy,    —(OCH₂CH₂)_(m)alkyl, cycloalkyl, heterocycloalkyl, aryl, or    heteroaryl, wherein the alkyl and alkoxy are each independently    optionally substituted with one or more substituents selected from    alkoxy, haloalkoxy, halogen, and —OH; and wherein the cycloalkyl,    heterocycloalkyl, aryl and heteroaryl are each independently    optionally substituted with one or more substituents selected from    alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —N₃, and —OH;-   R₁₀ is alkyl, alkenyl, alkynyl, —NR₇R₈, cycloalkyl,    heterocycloalkyl, aryl, amino, heteroalkyl, alkylamino, aminoalkyl    or heteroaryl, wherein the alkyl, alkenyl, and alkynyl are each    independently optionally substituted with one or more R₁₃; and    wherein the cycloalkyl, heterocycloalkyl, aryl and heteroaryl are    each independently optionally substituted with one or more R₁₂;-   each R₁₁ is independently at each occurrence alkyl, haloalkyl,    alkoxy, haloalkoxy, halogen, —NO₂, or —OH;-   each R₁₂ is independently at each occurrence aryl or heteroaryl,    wherein the aryl and heteroaryl are each independently optionally    substituted with one or more substituents selected from alkyl,    haloalkyl, alkoxy, haloalkoxy, halogen, and —OH;-   each R₁₃ is independently at each occurrence —OH, alkoxy,    heteroalkyl, aryloxy, —NH₂, arylalkyl, cycloalkyl, heterocycloalkyl,    aryl, heteroaryl, —O-aryl, —O-heteroaryl, —NR₇aryl, —NR₇heteroaryl,    or —NR₇C(═O)R₁₄, wherein the cycloalkyl, heterocycloalkyl, aryl,    heteroalkyl, and heteroaryl are each independently optionally    substituted with one or more substituents selected from alkyl,    haloalkyl, alkoxy, haloalkoxy, halogen, —NO₂, and —OH;-   R₁₄ is alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, cycloalkyl,    aryl, heterocyclyl, or heteroaryl, wherein the aryl and heteroaryl    are each independently optionally substituted with one or more R₁₅;    and wherein the alkyl, alkenyl, and alkynyl are each independently    optionally substituted with one or more substituents selected from    halogen and —OH;-   each R₁₅ is independently at each occurrence halogen, alkyl, CN,    —C(═O)alkyl, or —C(═O)alkenyl, wherein the alkyl and alkenyl is each    independently substituted with one or more substituents selected    from halogen and —OH;-   m is 1, 2, or 3; and-   n is 0 or 1;-   provided that:-   (i) if R₂ is —NO₂, —NHC(O)Me or —NH₂, and R₁, R₂, and R₄ are each H;    or R₁ is Me and R₂, R₃, and R₄ are each H; then R₆ is not —C(O)R₁₀    where R₁₀ is —(CH₂)—(CHMe)-phenyl;-   (ii) when R₂ is Cl, R₁, R₃ and R₄ are each H, R₆ is —C(═O)R₁₀, and    R₁₀ is (C₂-C₃)alkyl substituted with one R₁₃; then R₁₃ is not    unsubstituted cyclopentyl, unsubstituted phenyl or unsubstituted    2-thiophenyl; and-   (iii) when R₂ is Cl, and R₁, R₃ and R₄ are each H; then R₆ is    —C(═O)R₁₀, R₁₀ is not 1-ethylpropyl.    In some embodiments, the compound is a compound of Formula (Id):

or a pharmaceutically acceptable salt thereof, wherein:

-   R₁ is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NO₂,    —NH₂, CN, —NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈, wherein each    alkyl is independently optionally substituted with one or more R₉;-   R₂ is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NH₂,    —NO₂, CN, —NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈, wherein each    alkyl is independently optionally substituted with one or more R₉;-   R₃ is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NH₂,    —NO₂, CN, —NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈; wherein each    alkyl is independently optionally substituted with one or more R₉;-   R₄ is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NO₂,    —NH₂, CN, —NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈; wherein each    alkyl is independently optionally substituted with one or more R₉;    wherein R₁, R₂, R₃ and R₄ are not simultaneously H;-   R₅ is H, halogen, —CN, —OR₇, or —NR₇R₈;-   R₆ is alkyl, —C(═O)R₁₀, —C(═S)R₁₀, —C(O)NR₇R₈, cycloalkyl,    heterocycloalkyl, aryl, or heteroaryl, wherein the cycloalkyl,    heterocycloalkyl, aryl, heteroaryl are each independently optionally    substituted with one or more R₁₁; and wherein the alkyl is    substituted with one or more R₁₂;-   each R₇ and R₈ is independently H or alkyl;-   each R₉ is independently at each occurrence —NR₇R₈, alkoxy,    —(OCH₂CH₂)_(m)alkyl, cycloalkyl, heterocycloalkyl, aryl, or    heteroaryl, wherein the alkyl and alkoxy are each independently    optionally substituted with one or more substituents selected from    alkoxy, haloalkoxy, halogen, and —OH; and wherein the cycloalkyl,    heterocycloalkyl, aryl and heteroaryl are each independently    optionally substituted with one or more substituents selected from    alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —N₃, and —OH;-   R₁₀ is alkyl, alkenyl, alkynyl, —NR₇R₈, cycloalkyl,    heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, alkenyl,    and alkynyl are each independently optionally substituted with one    or more R₁₃; and wherein the cycloalkyl, heterocycloalkyl, aryl and    heteroaryl are each independently optionally substituted with one or    more R₁₂;-   each R₁₁ is independently at each occurrence alkyl, haloalkyl,    alkoxy, haloalkoxy, halogen, —NO₂, or —OH;-   each R₁₂ is independently at each occurrence aryl or heteroaryl,    wherein the aryl and heteroaryl are each independently optionally    substituted with one or more substituents selected from alkyl,    haloalkyl, alkoxy, haloalkoxy, halogen, and —OH;-   each R₁₃ is independently at each occurrence —OH, alkoxy, aryloxy,    —NH₂, arylalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,    —O-aryl, —O-heteroaryl, —NR₇aryl, —NR₇heteroaryl, or —NR₇C(═O)R₁₄,    wherein the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are    each independently optionally substituted with one or more    substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy,    halogen, —NO₂, and —OH;-   R₁₄ is alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, cycloalkyl,    aryl, heterocyclyl, or heteroaryl, wherein the aryl and heteroaryl    are each independently optionally substituted with one or more R₁₅;    and wherein the alkyl, alkenyl, and alkynyl are each independently    optionally substituted with one or more substituents selected from    halogen and —OH;    -   each R₁₅ is independently at each occurrence halogen, alkyl, CN,        —C(═O)alkyl, or —C(═O)alkenyl, wherein the alkyl and alkenyl is        each independently substituted with one or more substituents        selected from halogen and —OH;-   m is 1, 2, or 3; and-   n is 0 or 1;-   provided that:-   (i) if R₂ is —NO₂, —NHC(O)Me or —NH₂, and R₁, R₂, and R₄ are each H;    or R₁ is Me and R₂, R₃, and R₄ are each H; then R₆ is not —C(O)R₁₀    where R₁₀ is —(CH₂)—(CHMe)-phenyl;-   (ii) when R₂ is Cl, R₁, R₃ and R₄ are each H, R₆ is —C(═O)R₁₀, and    R₁₀ is (C₂-C₃)alkyl substituted with one R₁₃; then R₁₃ is not    unsubstituted cyclopentyl, unsubstituted phenyl or unsubstituted    2-thiophenyl; and-   (iii) when R₂ is Cl, and R₁, R₃ and R₄ are each H; then R₆ is    —C(═O)R₁₀, R₁₀ is not 1-ethylpropyl.

In some embodiments, R₁ is H, —NR₇C(═O)alkyl, or —NR₇R₈. In certainembodiments, R₁ is H. In some embodiments, R₃ is H, —NO₂, or —NR₇R₈. Incertain embodiments, R₃ is H. In some embodiments, R₄ is H. In someembodiments, each R₉ is independently at each occurrence —NR₇R₈, alkoxy,—(OCH₂CH₂)_(m)alkyl, heterocycloalkyl, or heteroaryl, wherein theheterocycloalkyl or heteroaryl are each independently optionallysubstituted with one or more substituents selected from alkyl, alkoxy,and —N₃. In some embodiments, n is 0, while in other embodiments, nis 1. In some embodiments, R₉ is heterocycloalkyl or heteroaryl. In someembodiments, R₉ is N-methylpiperazinyl, piperidinyl, or morpholinyl. Insome embodiments, R₉ is imidazolyl. In some embodiments, R₉ is azido. Insome embodiments, R₉ is —NR₇H. In some embodiments, R₇ is acyl,alkylacy, or alkenylacyl. In some embodiments, R₇ is

In some embodiments, R₂ is selected from halogen, —NH₂, —NO₂, CN,—NR₇C(═O)alkyl and —C(═O)NR₇alkyl, wherein each alkyl is independentlyoptionally substituted with one or more R₉. In certain embodiments, R₂is halo, such as chloro, fluoro or bromo. In some embodiments, R₂ ischloro. In some embodiments, R₂ is —NR₇C(═O)alkyl or —C(═O)NR₇alkyl, andthe alkyl is substituted with one R₉. In some embodiments, R₂ is nitroor —NHalkyl.

In some embodiments, R₅ is H, CN, —OH, or —NR₇R₈. In other embodiments,R₅ is —OH, or —NR₇R₈. In certain embodiments, R₅ is —OH, —NH₂, —N(H)CH₃or —N(CH₃)₂. In some embodiments, R₅ is —OH.

In some embodiments, R₆ is alkyl, —C(═O)R₁₀, —C(═S)R₁₀, aryl, orheteroaryl. In some embodiments, R₆ is —C(═O)R₁₀. In certainembodiments, R₁₀ is alkyl, alkenyl, alkynyl, —NR₇R₈, cycloalkyl, orheterocycloalkyl, each optionally substituted with one or more R₁₃. Inother embodiments, R₁₃ is independently at each occurrence —OH, alkoxy,aryloxy, —NH₂, arylalkyl, cycloalkyl, aryl, heteroaryl, or —NR₇C(═O)R₁₄.In some embodiments, R₁₄ is independently at each occurrence alkyl,haloalkyl, arylalkyl, alkenyl, heterocyclyl, or heteroaryl.

In some embodiments, R₁₀ is alkyl, alkenyl, amino, alkylamino, alkynyl,cycloalkyl, cycloalkyl, alkylamino, heteroaryl, or aminoalkyl. In someembodiments, the alkyl, amino, alkylamino, or cycloalkyl is substitutedwith aryl, aralkyl, heteroaryl, heterocyclyl, acylamino, aryloxy, orhydroxyl. In some embodiments, the aryl or heteroaryl is furthersubstituted with alkyl, halo, alkyloxy, or nitro. In some embodiments,the aryl or heteroaryl is further substituted with halo, alkyloxy, ornitro. In some embodiments, the acylamino is substituted with halo,alkenyl, heteroaryl, or heterocycloalkyl. In some embodiments, theheteroaryl or heterocycloalkyl is substituted with alkylacyl,alkenylacyl, or hydroxyl.

In some embodiments, R₂ is Cl, —NO₂, —NH₂ or —NR₇C(═O)alkyl, wherein thealkyl is optionally substituted with one or more R₉; R₆ is —C(═O)R₁₀;and R₁₀ is alkyl, alkenyl, alkynyl, cycloalkyl, and heterocycloalkyl,wherein the alkyl, alkenyl, and alkynyl are each optionally substitutedwith one or more R₁₃; and wherein the cycloalkyl and heterocycloalkylare each optionally substituted with one or more R₁₂

Also disclosed herein are compounds of Formula (Ib):

or a pharmaceutically acceptable salt thereof, wherein R₂, R₅ and R₆ areas defined above and herein.

Also disclosed herein are compounds of Formula (Ic):

or a pharmaceutically acceptable salt thereof, wherein variables R₅ andR₆ are as described above and herein.

In some embodiments, the compound of the invention is a compounddepicted in Table 3 or 4.

In certain embodiments, the present invention provides a pharmaceuticalpreparation suitable for use in a human patient, comprising any of thecompounds shown above (e.g., a compound of the invention, such as acompound of formula (I), and one or more pharmaceutically acceptableexcipients. In certain embodiments, the pharmaceutical preparations maybe for use in treating or preventing a condition or disease as describedherein. Any of the disclosed compounds may be used in the manufacture ofmedicaments for the treatment of any diseases or conditions disclosedherein.

The details of the disclosure are set forth in the accompanyingdescription below. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent disclosure, illustrative methods and materials are nowdescribed. Other features, objects, and advantages of the disclosurewill be apparent from the description and from the claims. In thespecification and the appended claims, the singular forms also includethe plural unless the context clearly dictates otherwise. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this disclosure belongs. All patents and publications cited inthis specification are incorporated herein by reference in theirentireties.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art ofthe present disclosure. The following references provide one of skillwith a general definition of many of the terms used in this disclosure:Singleton et al., Dictionary of Microbiology and Molecular Biology (2nded. 1994); The Cambridge Dictionary of Science and Technology (Walkered., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.),Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionaryof Biology (1991). As used herein, the following terms have the meaningsascribed to them below, unless specified otherwise.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

The term “and/or” is used in this disclosure to mean either “and” or“or” unless indicated otherwise.

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbylC(O)—, preferably alkyC(O)—.

The term “acylamino” is art-recognized and refers to an amino groupsubstituted with an acyl group and may be represented, for example, bythe formula hydrocarbylC(O)NH—.

The term “acyloxy” is art-recognized and refers to a group representedby the general formula hydrocarbylC(O)O—, preferably alkyC(O)O—.

The term “alkoxy” refers to an alkyl group, preferably a lower alkylgroup, having an oxygen attached thereto. Representative alkoxy groupsinclude methoxy, ethoxy, propoxy, tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl.

The term “alkenyl”, as used herein, refers to an aliphatic groupcontaining at least one double bond and is intended to include both“unsubstituted alkenyls” and “substituted alkenyls”, the latter of whichrefers to alkenyl moieties having substituents replacing a hydrogen onone or more carbons of the alkenyl group. Such substituents may occur onone or more carbons that are included or not included in one or moredouble bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed below, except where stability isprohibitive. For example, substitution of alkenyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

An “alkyl” group or “alkane” is a straight chained or branchednon-aromatic hydrocarbon which is completely saturated. Typically, astraight chained or branched alkyl group has from 1 to about 20 carbonatoms, preferably from 1 to about 10 unless otherwise defined. Examplesof straight chained and branched alkyl groups include methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl,pentyl and octyl. A C1-C6 straight chained or branched alkyl group isalso referred to as a “lower alkyl” group.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents, if nototherwise specified, can include, for example, a halogen, a hydroxyl, acarbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl),a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, asulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic orheteroaromatic moiety. It will be understood by those skilled in the artthat the moieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF3, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF3, —CN, and the like.

The term “Cx-y” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups that contain from x to y carbons in the chain. Forexample, the term “Cx-yalkyl” refers to substituted or unsubstitutedsaturated hydrocarbon groups, including straight-chain alkyl andbranched-chain alkyl groups that contain from x to y carbons in thechain, including haloalkyl groups such as trifluoromethyl and2,2,2-tirfluoroethyl, etc. C0 alkyl indicates a hydrogen where the groupis in a terminal position, a bond if internal. The terms “C2-yalkenyl”and “C2-yalkynyl” refer to substituted or unsubstituted unsaturatedaliphatic groups analogous in length and possible substitution to thealkyls described above, but that contain at least one double or triplebond respectively.

The term “heteroalkyl”, as used herein, refers to a saturated orunsaturated chain of carbon atoms and at least one heteroatom, whereinno two heteroatoms are adjacent.

Moreover, the term “heteroalkyl” (or “lower heteroalkyl”) as usedthroughout the specification, examples, and claims is intended toinclude both “unsubstituted heteroalkyl” and “substituted heteroalkyls”,the latter of which refers to heteroalkyl moieties having substituentsreplacing a hydrogen on one or more carbons or heteroatoms of thebackbone. Such substituents, if not otherwise specified, can include,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the heteroalkyl chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted heteroalkylmay include substituted and unsubstituted forms of amino, azido, imino,amido, phosphoryl (including phosphonate and phosphinate), sulfonyl(including sulfate, sulfonamido, sulfamoyl and sulfonate), and silylgroups, as well as ethers, alkylthios, carbonyls (including ketones,aldehydes, carboxylates, and esters), —CF3, —CN and the like.

The term “alkylamino”, as used herein, refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS—.

The term “alkynyl”, as used herein, refers to an aliphatic groupcontaining at least one triple bond and is intended to include both“unsubstituted alkynyls” and “substituted alkynyls”, the latter of whichrefers to alkynyl moieties having substituents replacing a hydrogen onone or more carbons of the alkynyl group. Such substituents may occur onone or more carbons that are included or not included in one or moretriple bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed above, except where stability isprohibitive. For example, substitution of alkynyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

The term “amide”, as used herein, refers to a group

wherein each R¹⁰ independently represents a hydrogen or hydrocarbylgroup, or two R¹⁰ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein each R¹⁰ independently represents a hydrogen or a hydrocarbylgroup, or two R¹⁰ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure. The term “aminoalkyl”, as used herein, refers to an alkylgroup substituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably, the ring is a 5- to 7-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The term “carbamate” is art-recognized and refers to a group

wherein R₉ and R₁₀ independently represent hydrogen or a hydrocarbylgroup, such as an alkyl group, or R₉ and R₁₀ taken together with theintervening atom(s) complete a heterocycle having from 4 to 8 atoms inthe ring structure.

The terms “carbocycle”, and “carbocyclic”, as used herein, refers to asaturated or unsaturated ring in which each atom of the ring is carbon.The term carbocycle includes both aromatic carbocycles and non-aromaticcarbocycles. Non-aromatic carbocycles include both cycloalkane rings, inwhich all carbon atoms are saturated, and cycloalkene rings, whichcontain at least one double bond.

The term “carbocycle” includes 5-7 membered monocyclic and 8-12 memberedbicyclic rings. Each ring of a bicyclic carbocycle may be selected fromsaturated, unsaturated and aromatic rings. Carbocycle includes bicyclicmolecules in which one, two or three or more atoms are shared betweenthe two rings. The term “fused carbocycle” refers to a bicycliccarbocycle in which each of the rings shares two adjacent atoms with theother ring. Each ring of a fused carbocycle may be selected fromsaturated, unsaturated and aromatic rings. In an exemplary embodiment,an aromatic ring, e.g., phenyl, may be fused to a saturated orunsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Anycombination of saturated, unsaturated and aromatic bicyclic rings, asvalence permits, is included in the definition of carbocyclic. Exemplary“carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane,1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene,bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fusedcarbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene,bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene andbicyclo[4.1.0]hept-3-ene. “Carbocycles” may be susbstituted at any oneor more positions capable of bearing a hydrogen atom.

A “cycloalkyl” group is a cyclic hydrocarbon which is completelysaturated. “Cycloalkyl” includes monocyclic and bicyclic rings.Typically, a monocyclic cycloalkyl group has from 3 to about 10 carbonatoms, more typically 3 to 8 carbon atoms unless otherwise defined. Thesecond ring of a bicyclic cycloalkyl may be selected from saturated,unsaturated and aromatic rings. Cycloalkyl includes bicyclic moleculesin which one, two or three or more atoms are shared between the tworings. The term “fused cycloalkyl” refers to a bicyclic cycloalkyl inwhich each of the rings shares two adjacent atoms with the other ring.The second ring of a fused bicyclic cycloalkyl may be selected fromsaturated, unsaturated and aromatic rings. A “cycloalkenyl” group is acyclic hydrocarbon containing one or more double bonds.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The term “carbonate” is art-recognized and refers to a group —OCO2-R10,wherein R10 represents a hydrocarbyl group.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO2H.

The term “ester”, as used herein, refers to a group —C(O)OR10 whereinR10 represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a hetaryl group.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heteroaryl” and “hetaryl” also include polycyclic ring systems havingtwo or more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heterocyclyl” and “heterocyclic” also include polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to behydrocarbyl for the purposes of this application, but substituents suchas acetyl (which has a ═O substituent on the linking carbon) and ethoxy(which is linked through oxygen, not carbon) are not. Hydrocarbyl groupsinclude, but are not limited to aryl, heteroaryl, carbocycle,heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl”, as used herein, refers to an alkyl groupsubstituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are ten or fewer non-hydrogen atoms in thesubstituent, preferably six or fewer. A “lower alkyl”, for example,refers to an alkyl group that contains ten or fewer carbon atoms,preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl,alkenyl, alkynyl, or alkoxy substituents defined herein are respectivelylower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, orlower alkoxy, whether they appear alone or in combination with othersubstituents, such as in the recitations hydroxyalkyl and aralkyl (inwhich case, for example, the atoms within the aryl group are not countedwhen counting the carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 5 to 7.

The term “silyl” refers to a silicon moiety with three hydrocarbylmoieties attached thereto.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that substituents canthemselves be substituted, if appropriate. Unless specifically stated as“unsubstituted,” references to chemical moieties herein are understoodto include substituted variants. For example, reference to an “aryl”group or moiety implicitly includes both substituted and unsubstitutedvariants.

The term “sulfate” is art-recognized and refers to the group —OSO3H, ora pharmaceutically acceptable salt thereof.

The term “sulfonamide” is art-recognized and refers to the grouprepresented by the general formulae

wherein R9 and R10 independently represents hydrogen or hydrocarbyl,such as alkyl, or R9 and R10 taken together with the intervening atom(s)complete a heterocycle having from 4 to 8 atoms in the ring structure.

The term “sulfoxide” is art-recognized and refers to the group—S(O)—R10, wherein R10 represents a hydrocarbyl.

The term “sulfonate” is art-recognized and refers to the group SO3H, ora pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group —S(O)2-R10,wherein R10 represents a hydrocarbyl.

The term “thioalkyl”, as used herein, refers to an alkyl groupsubstituted with a thiol group.

The term “thioester”, as used herein, refers to a group —C(O)SR10 or—SC(O)R10 wherein R10 represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

The term “urea” is art-recognized and may be represented by the generalformula

wherein R9 and R10 independently represent hydrogen or a hydrocarbyl,such as alkyl, or either occurrence of R9 taken together with R10 andthe intervening atom(s) complete a heterocycle having from 4 to 8 atomsin the ring structure.

The term “protecting group” refers to a group of atoms that, whenattached to a reactive functional group in a molecule, mask, reduce orprevent the reactivity of the functional group. Typically, a protectinggroup may be selectively removed as desired during the course of asynthesis. Examples of protecting groups can be found in Greene andWuts, Protective Groups in Organic Chemistry, 3^(rd) Ed., 1999, JohnWiley & Sons, NY and Harrison et al., Compendium of Synthetic OrganicMethods, Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representativenitrogen protecting groups include, but are not limited to, formyl,acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”),tert-butoxycarbonyl (“Boc”), trimethylsilyl (“TMS”),2-trimethylsilyl-ethanesulfonyl (“TES”), trityl and substituted tritylgroups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (“FMOC”),nitro-veratryloxycarbonyl (“NVOC”) and the like. Representative hydroxylprotecting groups include, but are not limited to, those where thehydroxyl group is either acylated (esterified) or alkylated such asbenzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranylethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers,such as ethylene glycol and propylene glycol derivatives and allylethers.

The term “prodrug” is intended to encompass compounds which, underphysiologic conditions, are converted into the therapeutically activeagents of the present invention (e.g., a compound of formula I). Acommon method for making a prodrug is to include one or more selectedmoieties which are hydrolyzed under physiologic conditions to reveal thedesired molecule. In other embodiments, the prodrug is converted by anenzymatic activity of the subject. For example, esters or carbonates(e.g., esters or carbonates of alcohols or carboxylic acids) arepreferred prodrugs of the present invention. In certain embodiments,some or all of the compounds of formula I in a formulation representedabove can be replaced with the corresponding suitable prodrug, e.g.,wherein a hydroxyl in the parent compound is presented as an ester or acarbonate or carboxylic acid present in the parent compound is presentedas an ester.

The present invention includes all pharmaceutically acceptableisotopically-labelled compounds as described herein wherein one or moreatoms are replaced by atoms having the same atomic number, but an atomicmass or mass number different from the atomic mass or mass numberusually found in nature. In certain embodiments, compounds of theinvention are enriched in such isotopically labeled substances (e.g.,compounds wherein the distribution of isotopes in the compounds in thecomposition differ from a natural or typical distribution of isotopes).

Examples of isotopes suitable for inclusion in the compounds of theinvention include isotopes of hydrogen, such as ²H and ³H carbon, suchas ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, such as ¹⁸F,iodine, such as ¹²³I and ¹²⁵I, nitrogen, such as ¹³N and ¹⁵N, oxygen,such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, and sulphur, such as³⁵S.

Certain isotopically-labelled compounds as disclosed herein, forexample, those incorporating a radioactive isotope, are useful in drugand/or substrate tissue distribution studies. The radioactive isotopestritium, i.e. ³H, and carbon-14, i.e. ¹⁴C, are useful for this purposein view of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron-emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Tomography (PET) studies forexamining substrate receptor occupancy.

Compounds of the invention can have one or more asymmetric carbon atomsand can exist in the form of optically pure enantiomers, mixtures ofenantiomers such as, for example, racemates, optically purediastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates. The optically activeforms can be obtained for example by resolution of the racemates, byasymmetric synthesis or asymmetric chromatography (chromatography with achiral adsorbents or eluant). That is, certain of the disclosedcompounds may exist in various stereoisomeric forms.

Stereoisomers are compounds that differ only in their spatialarrangement. Enantiomers are pairs of stereoisomers whose mirror imagesare not superimposable, most commonly because they contain anasymmetrically substituted carbon atom that acts as a chiral center.“Enantiomer” means one of a pair of molecules that are mirror images ofeach other and are not superimposable. “Diastereomers” are stereoisomersthat are not related as mirror images, most commonly because theycontain two or more asymmetrically substituted carbon atoms andrepresent the configuration of substituents around one or more chiralcarbon atoms. Enantiomers of a compound can be prepared, for example, byseparating an enantiomer from a racemate using one or more well-knowntechniques and methods, such as, for example, chiral chromatography andseparation methods based thereon. The appropriate technique and/ormethod for separating an enantiomer of a compound described herein froma racemic mixture can be readily determined by those of skill in theart.

“Geometric isomer” means isomers that differ in the orientation ofsubstituent atoms in relationship to a carbon-carbon double bond, to acycloalkyl ring, or to a bridged bicyclic system. Atoms (other than H)on each side of a carbon-carbon double bond may be in an E (substituentsare on opposite sides of the carbon-carbon double bond) or Z(substituents are oriented on the same side) configuration. “R,” “S,”“S*,” “R*,” “E,” “Z,” “cis,” and “trans,” indicate configurationsrelative to the core molecule. Certain of the disclosed compounds mayexist in atropisomeric forms. Atropisomers are stereoisomers resultingfrom hindered rotation about single bonds where the steric strainbarrier to rotation is high enough to allow for the isolation of theconformers. The compounds of the invention may be prepared as individualisomers by either isomer-specific synthesis or resolved from an isomericmixture. Conventional resolution techniques include forming the salt ofa free base of each isomer of an isomeric pair using an optically activeacid (followed by fractional crystallization and regeneration of thefree base), forming the salt of the acid form of each isomer of anisomeric pair using an optically active amine (followed by fractionalcrystallization and regeneration of the free acid), forming an ester oramide of each of the isomers of an isomeric pair using an optically pureacid, amine or alcohol (followed by chromatographic separation andremoval of the chiral auxiliary), or resolving an isomeric mixture ofeither a starting material or a final product using various well knownchromatographic methods.

Diastereomeric purity by weight is the ratio of the weight of onediastereomer or over the weight of all the diastereomers. When thestereochemistry of a disclosed compound is named or depicted bystructure, the named or depicted stereoisomer is at least about 60%,about 70%, about 80%, about 90%, about 99% or about 99.9% by weightrelative to the other stereoisomers. When a single enantiomer is namedor depicted by structure, the depicted or named enantiomer is at leastabout 60%, about 70%, about 80%, about 90%, about 99% or about 99.9% byweight optically pure. When a single diastereomer is named or depictedby structure, the depicted or named diastereomer is at least about 60%,about 70%, about 80%, about 90%, about 99% or about 99.9% by weightpure. Percent optical purity is the ratio of the weight of theenantiomer or over the weight of the enantiomer plus the weight of itsoptical isomer.

Percent purity by mole fraction is the ratio of the moles of theenantiomer (or diastereomer) or over the moles of the enantiomer (ordiastereomer) plus the moles of its optical isomer. When thestereochemistry of a disclosed compound is named or depicted bystructure, the named or depicted stereoisomer is at least about 60%,about 70%, about 80%, about 90%, about 99% or about 99.9% by molefraction pure relative to the other stereoisomers. When a singleenantiomer is named or depicted by structure, the depicted or namedenantiomer is at least about 60%, about 70%, about 80%, about 90%, about99% or about 99.9% by mole fraction pure. When a single diastereomer isnamed or depicted by structure, the depicted or named diastereomer is atleast about 60%, about 70%, about 80%, about 90%, about 99% or about99.9% by mole fraction pure.

When a disclosed compound is named or depicted by structure withoutindicating the stereochemistry, and the compound has at least one chiralcenter, it is to be understood that the name or structure encompasseseither enantiomer of the compound free from the corresponding opticalisomer, a racemic mixture of the compound or mixtures enriched in oneenantiomer relative to its corresponding optical isomer. When adisclosed compound is named or depicted by structure without indicatingthe stereochemistry and has two or more chiral centers, it is to beunderstood that the name or structure encompasses a diastereomer free ofother diastereomers, a number of diastereomers free from otherdiastereomeric pairs, mixtures of diastereomers, mixtures ofdiastereomeric pairs, mixtures of diastereomers in which onediastereomer is enriched relative to the other diastereomer(s) ormixtures of diastereomers in which one or more diastereomer is enrichedrelative to the other diastereomers. The invention embraces all of theseforms.

As used herein, the term “pharmaceutically acceptable salt” means anypharmaceutically acceptable salt of the compound of formula (I). Forexample, pharmaceutically acceptable salts of any of the compoundsdescribed herein include those that are within the scope of soundmedical judgment, suitable for use in contact with the tissues of humansand animals without undue toxicity, irritation, allergic response andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example,pharmaceutically acceptable salts are described in: Berge et al., J.Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts:Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth),Wiley-VCH, 2008. The salts can be prepared in situ during the finalisolation and purification of the compounds described herein orseparately by reacting a free base group with a suitable organic acid.

The compounds of the invention may have ionizable groups so as to becapable of preparation as pharmaceutically acceptable salts. These saltsmay be acid addition salts involving inorganic or organic acids or thesalts may, in the case of acidic forms of the compounds of the inventionbe prepared from inorganic or organic bases. Frequently, the compoundsare prepared or used as pharmaceutically acceptable salts prepared asaddition products of pharmaceutically acceptable acids or bases.Suitable pharmaceutically acceptable acids and bases and methods forpreparation of the appropriate salts are well-known in the art. Saltsmay be prepared from pharmaceutically acceptable non-toxic acids andbases including inorganic and organic acids and bases.

Representative acid addition salts include acetate, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, and valeratesalts. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, and magnesium, as well as nontoxicammonium, quaternary ammonium, and amine cations, including, but notlimited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, andethylamine.

The term “subject” to which administration is contemplated includes, butis not limited to, humans (i.e., a male or female of any age group,e.g., a pediatric subject (e.g., infant, child, adolescent) or adultsubject (e.g., young adult, middle-aged adult or senior adult)) and/orother primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals,including commercially relevant mammals such as cattle, pigs, horses,sheep, goats, cats, and/or dogs; and/or birds, including commerciallyrelevant birds such as chickens, ducks, geese, quail, and/or turkeys.Preferred subjects are humans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample.

In treatment, the object is to prevent or slow down (lessen) anundesired physiological condition, disorder, or disease, or obtainbeneficial or desired clinical results. Beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms;diminishment of the extent of a condition, disorder, or disease;stabilized (i.e., not worsening) state of condition, disorder, ordisease; delay in onset or slowing of condition, disorder, or diseaseprogression; amelioration of the condition, disorder, or disease stateor remission (whether partial or total), whether detectable orundetectable; an amelioration of at least one measurable physicalparameter, not necessarily discernible by the patient; or enhancement orimprovement of condition, disorder, or disease. Treatment includeseliciting a clinically significant response without excessive levels ofside effects. Treatment also includes prolonging survival as compared toexpected survival if not receiving treatment.

Methods of Use

Ubiquitin is a 76-amino acid protein attached to substrate proteinspost-translationally via iso-peptide bond formation between ubiquitin'sC-terminal glycine and a substrate lysine sidechain; linear and branchedpolyubiquitin chains are assembled via attachment of another molecule ofubiquitin to one of seven lysines or the N-terminal methionine ofubiquitin. (Pickart and Fushman, Curr Opin Chem Biol, 8, 610-6, 2004)Ubiquitin is attached to substrate proteins by the coordinated action ofubiquitin activating (E1), conjugating (E2), and ligating (E3) enzymesand removed by a family of proteases known as deubiquitinating enzymes(DUBs). The first recognized role of the ubiquitin system wascontrolling protein turnover. (Lecker et al., J Am Soc Nephrol, 17,1807-19, 2006) Ubiquitin tags are also responsible for signaling a widerange of non-degradative functions. (O'Neill, J Biol Chem, 284, 8209,2009) Ubiquitination can affect protein activity by modulatingconformational changes, complexation with other proteins, susceptibilityto addition of other post-translation modifications (PTM) includingphosphorylation and acetylation, and cellular localization. Throughcombined degradative and non-degradative functions, ubiquitinationcoordinates a wide range of cellular processes including proteolysis,DNA repair, chromatin remodeling, receptor signaling, and immunity,among others. (Pinto-Fernandez and Kessler, Front Genet, 7, 133, 2016)Not surprisingly, aberrant ubiquitin system activity is linked todisease, most heavily cancer, infection, and neurodegeneration. (D'Arcyet al., Pharmacol Ther, 147, 32-54, 2015; Atkin and Paulson, Front MolNeurosci, 7, 63, 2014; Nanduri et al., Curr Pharm Des, 19, 3234-47,2013) Deregulation of the ubiquitin-proteasome system has beenimplicated in the pathogenesis of many humandiseases, including cancer(Hoeller et al. Nat Rev Cancer 2006, 6(10), 776-788), neurodegenerativedisorders (Rubinsztein, Nature 2006, 443(7113), 780-786) and viraldiseases (Gao & Luo Can J Physiol Pharmacol 2006, 84(1), 5-14). Therelationship between ubiquitin and cancer biology has been clinicallyvalidated by the FDA approval of the proteasome inhibitor bortezomib formultiple myeloma. (Kane et al., Oncologist, 8, 508-13, 2003)

There are approximately 100 human DUBs belonging to six distinctfamilies. Five of the families [ubiquitin specific protease (USP),ubiquitin C-terminal hydrolase (UCH), Ovarian tumor protease (OTU),Josephin, and Mindy] are cysteine proteases and the sixth [JAB/MPN/MOV34(JAMM/MPN)] is comprised of zinc metalloproteases. (Komander et al., NatRev Mol Cell Biol, 10, 550-63, 2009; Clague et al., Physiol Rev, 93,1289-315, 2013; Abdul Rehman et al., Mol Cell, 63, 146-55, 2016) ManyDUBs have been linked to physiological and/or pathophysiologicalfunctions. Ubiquitin specific proteases and ubiquitin C-terminalhydrolases (UCH) enzymes are the best characterized members of the DUBfamily (Komander et al. 2009; Nijman et al. Cell 2005, 123(5), 773-786).For example, USP1 and USP4 are involved in DNA damage repair. (Kee andHuang, Mol Cell Biol, 36, 524-44, 2015) USP22 and BAP1 have a role inchromatin function (Atanassov et al., FEBS Lett, 585, 2016-23, 2011),and USP2 and USP8 are reported to stabilize oncogenic proteins cyclinD1, (Shan et al., 2009) and mutant EGFR, (Byun et al., Clin Cancer Res,19, 3894-904, 2013) respectively. X-ray crystal structures of thecatalytic core of each family reveal that all except the Mindy familyadopt a common fold comprised of three domains: the fingers domaincoordinates the ubiquitin core, and the thumb and palm coordinates theubiquitin tail at the catalytic triad-containing active site. (Komanderet al., 2009) While dozens of apo- and ubiquitin-bound structures havebeen solved, very few have been achieved with non-ubiquitin-basedcompounds. Notably, there are no reported small molecule DUB complexstructures for the largest 56-member mammalian USP family. (Komander etal., 2009), (Davies et al., Bioorg Med Chem Lett, 22, 3900-4, 2012;Ratia et al., Proc Natl Acad Sci USA, 105, 16119-24, 2008; Schlierf etal., Nat Commun, 7, 13166, 2016)

Although DUBs are generally regarded as a targetable class for drugdevelopment, inhibitor development is still in early stages. The firstDUB inhibitor, the dual USP14/UCHL5 inhibitor VLX1570, entered clinicaltrials in 2015. (Wang et al., Sci Rep, 6, 26979, 2016b) The only exampleof structure-guided development of a DUB inhibitor, which targeted theSARs DUB PLPro (Baez-Santos et al., Antiviral Res, 115, 21-38, 2015),generated compounds with IC₅₀s below 500 nM and exhibiting a high degreeof selectivity relative to mammalian DUBs. In this case, selectivity mayresult from significant structural differences between viral andmammalian DUBs. There are, however, no reported examples ofstructure-guided optimization of a mammalian DUB. However, breakthroughsin X-ray crystallography of small molecule DUB inhibitor complexes hasthe potential to enable rapid development of potent and selectiveinhibitors.

USP7 (Ubiquitin Specific Protease 7)/HAUSP (Herpes Associated UbiquitinSpecific Protease) is a 135 kDa protein of the USP family. USP7 has beenshown to interact with viral proteins, such as ICPO (Vmw 110), a herpessimplex virus immediate-early gene stimulating initiation of the virallytic cycle (Everett et al., J Virol 73, 1999, 417-426), and EBNA1(Epstein-Barr Nuclear Antigen-1) (Holowaty et al., J Biol Chem 2003,278, 29987-29994 and 47753-47761). The DUB USP7 has been shown to beinvolved in regulation of a myriad of cellular processes, includingepigenetics, cell cycle, DNA repair, immunity, viral infection andtumorigenesis. Interest in the enzyme intensified when USP7 wasimplicated in regulating degradation of the tumor suppressor p53 (Li etal., Nature, 416, 648-53, 2002), by stabilizing the major E3 ligase forp53, MDM2. (Cummins et al., Nature, 428, 1 p following 486, 2004, Li etal., Mol Cell, 13, 879-86, 2004). Consistent with recent reports, USP7silencing has also been shown to increase steady-state p53 levels bypromoting Mdm2 degradation. Binding of USP7 to p53 was recently shown tobe regulated by TSPYL5, a protein potentially involved in breastoncogenesis through a competition with p53 for binding to the sameregion of USP7. (Epping et al., Nat Cell Biol. 2011, 13(1):102-8) Morerecently, both upregulation and downregulation of USP7 have been shownto inhibit colon cancer cell proliferation in vitro and tumor growth invivo, by resulting in constitutively high p53 levels (Becker et al. CellCycle 2008, 7(9), 1205-13).

USP7 also alters the level of the p16_(INK4a) tumor suppressor throughBmi1/Mel18 stabilization (Maertens et al., Embo J. 2010 29, 2553-2565).Additional proteins involved in genomic integrity/regulation such as theDNMT1 DNA methylase and the Claspin adaptor are also stabilized by USP7(Du et al., Science Signaling 2010, 3(146):ra80; Faustrup et al., J.Cell Biol. 2009, 184(1):13-9). Importantly, the abundance of USP7 andDNMT1, a protein involved in maintaining epigenetic methylation requiredto silence genes involved in development and cancer, correlates in humancolon cancer (Du et al., 2010). USP7 has also been shown in human cellsto deubiquitinate the well-known tumor suppressor gene PTEN, whichprovokes its nuclear export and hence its inactivation (Song et al.,Nature 2008, 455(7214), 813-7). More importantly, USP7 overexpressionwas reported for the first time in prostate cancer and thisoverexpression was directly associated with tumour aggressiveness (Songet al., Nature 2008, 455(7214), 813-7).

Recently, several epigenetic modifiers, including the methytransferasePHF8, (Wang et al., 2016a) demethylase DNMT1, (Du et al., 2010, Felle etal., Nucleic Acids Res, 39, 8355-65, 2011, Qin et al., J Cell Biochem,112, 439-44, 2011) and acetyltransferase Tip60, (Dar et al., Mol CellBiol, 33, 3309-20, 2013) as well as H2B itself, (van der Knaap et al.,Mol Cell, 17, 695-707, 2005) have been identified as direct targets ofUSP7. Other notable targets of USP7 include the transcription factorsFOXP3, which in Treg cells links this DUB enzyme to immune response,(van Loosdregt et al., Immunity, 39, 259-71, 2013) and N-Myc, which isstabilized in neuroblastoma cells. (Tavana et al., Nat Med, 22,1180-1186, 2016) Consistent with its regulation of diverse substratesand biological processes USP7 has emerged as a drug target in a widerange of malignancies including multiple myeloma, (Chauhan et al.,Cancer Cell, 22, 345-58, 2012) breast cancer, (Wang et al., 2016a)neuroblastoma, (Tavana et al., 2016)glioma, (Cheng et al., Oncol Rep,29, 1730-6, 2013) and ovarian cancer. (Zhang et al., Tohoku J Exp Med,239, 165-75, 2016) USP7 has also been shown in human cells todeubiquitinate FOXO4, which provokes its nuclear export and hence itsinactivation; consequently the oncogenic PI3K/PKB signaling pathway wasactivated (van der Horst et al., Nat Cell Biol. 2006, 8, 1064-1073)Finally, USP7 plays an important role in p53-mediated cellular responsesto various types of stress, such as DNA damage and oxidative stress(Marchenko et al., Embo J. 2007 26, 923-934, Meulmeester et al., MolCell 2005, 18, 565-576, van der Horst et al., Nat Cell Biol. 2006, 8,1064-1073).

Multiple myeloma (MMz) is an incurable hematological malignancycharacterized by the accumulation of abnormal plasma cells in the bonemarrow. which impede production of normal blood cells. the averagesurvival of MM patients has improved in recent years as a result of theintroduction of proteasome inhibitors and immunomodulatory agents intotreatment regimens but is still quite poor at only 5 years. Theproteasome inhibitor bortezomiib validates the ubiquitin proteasomesystem as a therapeutic target for MM drug development. USP7 is atherapeutic target in MM due to its role in the degradation of p53. USP7is highly expressed in MM patient tumor cells and MM cell lines versusnormal bone marrow cells. Mutations or deletions in p53 are late eventsin MM suggesting that increasing p53via pharmacological inhibition ofUSP7 could be an effective therapeutic strategy for this malignancy.

P22077 and its close analog P5091 (structures in FIG. 1A) are theinhibitors most frequently utilized to probe USP7 functions. P22077exhibits modest potency against USP7 (IC50=8.0 μM) and equipotentinhibition of two additional DUBs, USP10 and USP47. (Altun et al., 2011,Ritorto et al., 2014) In addition to modest potency and selectivity,reported drawbacks of these nitro-thiophene-based compounds include poorsolubility and general toxicity. (Chen et al., 2017) Additional USP7inhibitors (shown in FIG. 1B) have been identified although none possessfeatures superior to P5091/P22077 and significant optimization effortshave not been undertaken. (Reverdy et al., Chem Biol, 19, 467-77, 2012;Colland et al., Mol Cancer Ther, 8, 2286-95, 2009; Aleo et al., CancerRes, 66, 9235-44, 2006; Nicholson et al., Protein Sci, 17, 1035-43,2008, Yamaguchi et al., Bioorg Med Chem Lett, 23, 3884-6, 2013,Tanokashira et al., Tetrahedron, 72, 5530-5540, 2016.

Disclosed herein are methods for treating and preventing diseases andconditions that benefit from the modulation of USP7, comprisingadministering to a subject in need thereof a compound of Formula (I) ora pharmaceutically acceptable salt thereof.

Disclosed herein are methods for treating and preventing diseases andconditions that benefit from the inhibition of USP7, comprisingadministering to a subject in need thereof a compound of Formula (I) ora pharmaceutically acceptable salt thereof.

Disclosed herein are methods of inhibiting USP7, comprisingadministering to a subject in need thereof a compound of Formula (I) ora pharmaceutically acceptable salt thereof.

In certain embodiments, disclosed herein are methods of treating adisease or a disorder modulated by USP7 comprising administering to asubject in need thereof a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof. In certain embodiments, disclosed herein aremethods of preventing a disease or a disorder modulated by USP7comprising administering to a subject in need thereof a compound ofFormula (I) or a pharmaceutically acceptable salt thereof. In someembodiments, the modulation of USP7 involves inhibiting USP7.

In one embodiment, the disease or disorder is selected from cancer andmetastasis, neurodegenerative diseases, immunological disorders,diabetes, bone and joint diseases, osteoporosis, arthritis inflammatorydisorders, cardiovascular diseases, ischemic diseases, viral infectionsand diseases, viral infectivity and/or latency, and bacterial infectionsand diseases.

Disclosed herein is the use of an inhibitor of USP7 for the preparationof a medicament for treating or preventing a disease or conditionmodulated by USP7, wherein the medicament comprises a compound ofFormula (I). In some embodiments, the modulation of USP7 involvesinhibiting USP7.

Disclosed herein a compound of Formula (I) for use in treating a diseaseor condition modulated by USP7. In some embodiments, the modulation ofUSP7 involves inhibiting USP7.

Disclosed herein are methods of treating cancer comprising administeringto a subject in need thereof a compound of Formula (I) or apharmaceutically acceptable salt thereof.

In some embodiments, exemplary cancers include, but are not limited to,liposarcoma, neuroblastoma, glioblastoma, breast cancer, bladder cancer,glioma, neuoblastoma, adrenocortical cancer, multiple myeloma,colorectal cancer, non-small cell lung cancer, Human PapillomaVirus-associated cervical, oropharyngeal, penis, ovarian cancer, anal,thyroid or vaginal cancer or Epstein-Barr Virus-associatednasopharyngeal carcinoma, gastric cancer, rectal cancer, thyroid cancer,Hodgkin lymphoma or diffuse large B-cell lymphoma.

In some embodiments, the cancers are selected from multiple myeloma,breast cancer, neuroblastoma, glioma, colon cancer, prostate cancer,neuroblastoma, and ovarian cancer. In some embodiments, the cancer isbreast cancer, glioma, neuoblastoma, multiple myeloma, or ovariancancer. In some embodiments, the cancer is multiple myeloma.

Disclosed herein are methods of treating neurodegenerative diseasescomprising administering to a subject in need thereof a compound ofFormula (I) or a pharmaceutically acceptable salt thereof.

In some embodiments, neurodegenerative diseases include, but are notlimited to, Alzheimer's disease, multiple sclerosis, Huntington'sdisease, infectious meningitis, encephalomyelitis, Parkinson's disease,amyotrophic lateral sclerosis, or encephalitis.

Pharmaceutical Compositions

The compositions and methods of the present invention may be utilized totreat a subject in need thereof. In certain embodiments, the subject isa mammal such as a human, or a non-human mammal. When administered tosubject, such as a human, the composition or the compound is preferablyadministered as a pharmaceutical composition comprising, for example, acompound of the invention and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are well known in the art andinclude, for example, aqueous solutions such as water or physiologicallybuffered saline or other solvents or vehicles such as glycols, glycerol,oils such as olive oil, or injectable organic esters. In a preferredembodiment, when such pharmaceutical compositions are for humanadministration, particularly for invasive routes of administration(i.e., routes, such as injection or implantation, that circumventtransport or diffusion through an epithelial barrier), the aqueoussolution is pyrogen-free, or substantially pyrogen-free. The excipientscan be chosen, for example, to effect delayed release of an agent or toselectively target one or more cells, tissues or organs. Thepharmaceutical composition can be in dosage unit form such as tablet,capsule (including sprinkle capsule and gelatin capsule), granule,lyophile for reconstitution, powder, solution, syrup, suppository,injection or the like. The composition can also be present in atransdermal delivery system, e.g., a skin patch. The composition canalso be present in a solution suitable for topical administration, suchas an eye drop.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable agents that act, for example, to stabilize, increasesolubility or to increase the absorption of a compound such as acompound of the invention. Such physiologically acceptable agentsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. The choice of a pharmaceutically acceptable carrier,including a physiologically acceptable agent, depends, for example, onthe route of administration of the composition. The preparation orpharmaceutical composition can be a self-emulsifying drug deliverysystem or a self-microemulsifying drug delivery system. Thepharmaceutical composition (preparation) also can be a liposome or otherpolymer matrix, which can have incorporated therein, for example, acompound of the invention. Liposomes, for example, which comprisephospholipids or other lipids, are nontoxic, physiologically acceptableand metabolizable carriers that are relatively simple to make andadminister.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of a subject without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

A pharmaceutical composition (preparation) can be administered to asubject by any of a number of routes of administration including, forexample, orally (for example, drenches as in aqueous or non-aqueoussolutions or suspensions, tablets, capsules (including sprinkle capsulesand gelatin capsules), boluses, powders, granules, pastes forapplication to the tongue); absorption through the oral mucosa (e.g.,sublingually); anally, rectally or vaginally (for example, as a pessary,cream or foam); parenterally (including intramuscularly, intravenously,subcutaneously or intrathecally as, for example, a sterile solution orsuspension); nasally; intraperitoneally; subcutaneously; transdermally(for example as a patch applied to the skin); and topically (forexample, as a cream, ointment or spray applied to the skin, or as an eyedrop). The compound may also be formulated for inhalation. In certainembodiments, a compound may be simply dissolved or suspended in sterilewater. Details of appropriate routes of administration and compositionssuitable for same can be found in, for example, U.S. Pat. Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, the particular mode of administration. The amountof active ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an active compound, such as a compound ofthe invention, with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules (including sprinkle capsules and gelatin capsules),cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), lyophile, powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a compound of the present invention as anactive ingredient. Compositions or compounds may also be administered asa bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules(including sprinkle capsules and gelatin capsules), tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; (10) complexing agents,such as, modified and unmodified cyclodextrins; and (11) coloringagents. In the case of capsules (including sprinkle capsules and gelatincapsules), tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions, such as dragees, capsules (including sprinkle capsules andgelatin capsules), pills and granules, may optionally be scored orprepared with coatings and shells, such as enteric coatings and othercoatings well known in the pharmaceutical-formulating art. They may alsobe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms useful for oral administration includepharmaceutically acceptable emulsions, lyophiles for reconstitution,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, cyclodextrins and derivatives thereof, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions for rectal, vaginal, orurethral administration may be presented as a suppository, which may beprepared by mixing one or more active compounds with one or moresuitable nonirritating excipients or carriers comprising, for example,cocoa butter, polyethylene glycol, a suppository wax or a salicylate,and which is solid at room temperature, but liquid at body temperatureand, therefore, will melt in the rectum or vaginal cavity and releasethe active compound.

Formulations of the pharmaceutical compositions for administration tothe mouth may be presented as a mouthwash, or an oral spray, or an oralointment.

Alternatively or additionally, compositions can be formulated fordelivery via a catheter, stent, wire, or other intraluminal device.Delivery via such devices may be especially useful for delivery to thebladder, urethra, ureter, rectum, or intestine.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane. Transdermal patches have the added advantage ofproviding controlled delivery of a compound of the present invention tothe body. Such dosage forms can be made by dissolving or dispersing theactive compound in the proper medium. Absorption enhancers can also beused to increase the flux of the compound across the skin. The rate ofsuch flux can be controlled by either providing a rate controllingmembrane or dispersing the compound in a polymer matrix or gel.Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.Exemplary ophthalmic formulations are described in U.S. Publication Nos.2005/0080056, 2005/0059744, 2005/0031697 and 2005/004074 and U.S. Pat.No. 6,583,124, the contents of which are incorporated herein byreference. If desired, liquid ophthalmic formulations have propertiessimilar to that of lacrimal fluids, aqueous humor or vitreous humor orare compatible with such fluids. A preferred route of administration islocal administration (e.g., topical administration, such as eye drops,or administration via an implant).

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

Pharmaceutical compositions suitable for parenteral administrationcomprise one or more active compounds in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

For use in the methods of this invention, active compounds can be givenper se or as a pharmaceutical composition containing, for example, 0.1to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinaceous biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a compound at a particular targetsite.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the subject being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the concentration of a compound that is sufficient to elicit thedesired therapeutic effect. It is generally understood that theeffective amount of the compound will vary according to the weight, sex,age, and medical history of the subject. Other factors which influencethe effective amount may include, but are not limited to, the severityof the subject's condition, the disorder being treated, the stability ofthe compound, and, if desired, another type of therapeutic agent beingadministered with the compound of the invention. A larger total dose canbe delivered by multiple administrations of the agent. Methods todetermine efficacy and dosage are known to those skilled in the art(Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13ed., 1814-1882, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in thecompositions and methods of the invention will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentinvention, the active compound may be administered two or three timesdaily. In preferred embodiments, the active compound will beadministered once daily.

Effective dosage amounts of the disclosed compounds, when used for theindicated effects, range from about 0.5 mg to about 5000 mg of thedisclosed compound as needed to treat the condition. Compositions for invivo or in vitro use can contain about 0.5, about 5, about 20, about 50,about 75, about 100, about 150, about 250, about 500, about 750, about1000, about 1250, about 2500, about 3500, or about 5000 mg of thedisclosed compound, or, in a range of from one amount to another amountin the list of doses

In certain embodiments, compounds of the invention may be used alone orconjointly administered with another type of therapeutic agent. As usedherein, the phrase “conjoint administration” refers to any form ofadministration of two or more different therapeutic compounds such thatthe second compound is administered while the previously administeredtherapeutic compound is still effective in the body (e.g., the twocompounds are simultaneously effective in the subject, which may includesynergistic effects of the two compounds). For example, the differenttherapeutic compounds can be administered either in the same formulationor in a separate formulation, either concomitantly or sequentially. Incertain embodiments, the different therapeutic compounds can beadministered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72hours, or a week of one another. Thus, a subject who receives suchtreatment can benefit from a combined effect of different therapeuticcompounds.

In certain embodiments, conjoint administration of compounds of theinvention with one or more additional therapeutic agent(s) providesimproved efficacy relative to each individual administration of thecompound of the invention (e.g., compound of formula I or Ia) or the oneor more additional therapeutic agent(s). In certain such embodiments,the conjoint administration provides an additive effect, wherein anadditive effect refers to the sum of each of the effects of individualadministration of the compound of the invention and the one or moreadditional therapeutic agent(s).

This invention includes the use of pharmaceutically acceptable salts ofcompounds of the invention in the compositions and methods of thepresent invention. In certain embodiments, contemplated salts of theinvention include, but are not limited to, alkyl, dialkyl, trialkyl ortetra-alkyl ammonium salts. In certain embodiments, contemplated saltsof the invention include, but are not limited to, L-arginine,benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol,diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine,ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium,L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine,potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine,tromethamine, and zinc salts. In certain embodiments, contemplated saltsof the invention include, but are not limited to, Na, Ca, K, Mg, Zn orother metal salts.

The pharmaceutically acceptable acid addition salts can also exist asvarious solvates, such as with water, methanol, ethanol,dimethylformamide, and the like. Mixtures of such solvates can also beprepared. The source of such solvate can be from the solvent ofcrystallization, inherent in the solvent of preparation orcrystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

EXAMPLES

The compounds of Formula (I) may be prepared by methods known in the artof organic synthesis as set forth in part by the following syntheticschemes. The compounds described herein may be made from commerciallyavailable starting materials or synthesized using known organic,inorganic, and/or enzymatic processes.

In the schemes described below, it is well understood that protectinggroups for sensitive or reactive groups are employed where necessary inaccordance with general principles or chemistry. Protecting groups aremanipulated according to standard methods of organic synthesis (T. W.Greene and P. G. M. Wuts, “Protective Groups in Organic Synthesis”,Third edition, Wiley, New York 1999). These groups are removed at aconvenient stage of the compound synthesis using methods that arereadily apparent to those skilled in the art. The selection processes,as well as the reaction conditions and order of their execution, shallbe consistent with the preparation of compounds of Formula (I).

Those skilled in the art will recognize if a stereocenter exists in thecompounds of Formula (I). Accordingly, the present disclosure includesboth possible stereoisomers (unless specified in the synthesis) andincludes not only racemic compounds but the individual enantiomersand/or diastereomers as well. When a compound is desired as a singleenantiomer or diastereomer, it may be obtained by stereospecificsynthesis or by resolution of the final product or any convenientintermediate. Resolution of the final product, an intermediate, or astarting material may be affected by any suitable method known in theart. See, for example, “Stereochemistry of Organic Compounds” by E. L.Eliel, S. H. Wilen, and L. N. Mander (Wiley-Interscience, 1994). Amixture of enantiomers, diastereomers, cis/trans isomers resulting fromthe process described above can be separated into their singlecomponents by chiral salt technique, chromatography using normal phase,reverse phase or chiral column, depending on the nature of theseparation.

The disclosure is further illustrated by the following examples andsynthesis schemes, which are not to be construed as limiting thisdisclosure in scope or spirit to the specific procedures hereindescribed. It is to be understood that the examples are provided toillustrate certain embodiments and that no limitation to the scope ofthe disclosure is intended thereby. It is to be further understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which may suggest themselves to those skilled in theart without departing from the spirit of the present disclosure and/orscope of the appended claims.

Analytical Methods, Materials, and Instrumentation

Unless otherwise noted, reagents and solvents were used as received fromcommercial suppliers. All commercially available starting materials werepurchased from Sigma Aldrich, Fisher Scientific, Oakwood Chemical andCombi Block. All reagents were used as received without furtherpurification. Known compounds were synthesized according to publishedliterature procedures and any modifications are noted. Anhydroussolvents, such as tetrahydrofuran (THF), diethyl ether, dichloromethane(DCM), dimethyl formamide (DMF), dimethylsulfoxide (DMSO), 1,4-dioxane,and toluene (PhMe) were purchased from Fisher Scientific, and used asreceived. If necessary, air or moisture sensitive reactions were carriedout under an inert atmosphere of nitrogen.

Removal of solvents was accomplished on a Buchi R-300 rotary evaporatorand further concentration was done under a Welch 1400B-01 vacuum line,and Labconco FreeZone 6 plus system. Purification of compounds wasperformed by normal phase column chromatography using TeledyneCombiFlash chromatography system, and/or reversed phase chromatographyon Waters Micromass ZQ preparative system with SunFire® Prep C18 OBD™ 5μM column. The purity was analyzed on Waters Acquity UPLC system.Analytical thin layer chromatography (TLC) plates were purchased fromFisher Scientific (EMD Millipore TLC Silica Gel60 F254). Visualizationwas accomplished by irradiation under UV light (254 nm).

All 1H-NMR spectra were recorded at 298K on a Bruker ARX 500 (500 MHz)spectrometer. 13C-NMR spectra were recorded on a Bruker ARX 500 (126MHz) spectrometer. Samples were dissolved in CDCl3, DMSO-d6, or CD3OD.The spectra were referenced to the residual solvent peak (chlorofrom-d:7.26 ppm for 1H-NMR and 77.16 ppm for 13C-NMR; DMSO-d6: 2.50 ppm for1H-NMR and 39.25 ppm for 13C-NMR, CD3OD: 3.31 ppm for 1H NMR and 49.00ppm for 13C NMR or tetramethylsilane (TMS) as the internal standard.Chemical shift, multiplicity (s=singlet, d=doublet, t=triplet,q=quartet, m=multiplet, br-broad peak), coupling constants (Hz), andnumber of protons. Mass spectrometry (LCMS) data were obtained on WatersAcquity UPLC system in positive ESI mode.

Example 1: Exemplary Synthesis of Compounds of the Disclosure

2-Aminobenzoic acids (10.0 mmol) and formamide (1.8 g, 40.0 mmol) weremixed in pressure tube, which was heated at 150° C. overnight. Then thereaction was cooled to room temperature. The solid was suspended in coldwater, then collected by vacuum filtration, and dried on high vacuumline. The products 1.6 g (—Cl) and 1.8 g (—NO₂) were isolated as lightbrown solid in 88% (—Cl) and 95% (—NO₂) yields with no furtherpurification.

Sodium hydride (60% dispersion in mineral oil) (0.88 g, 22.0 mmol) wasdissolved in 40 mL anhydrous DMSO at 0° C. under N₂.Trimethylsulfoxonium iodide (4.84 g, 22.0 mmol) was added into thesolution portionwise. When addition completed, the mixture was warmed upto room temperature, and stirred for 40 min. Then 1-Boc-4-piperidone(3.98 g, 20.0 mmol) was added portion wisely. The reaction mixture wasthen stirred at room temperature for 1 hour, then at 65° C. for anotherhour. Then the mixture was poured on 100 mL ice. Aqueous phase wasextracted using EtOAc (50 mL×2). Combined organic phase was washed withbrine, dried over MgSO₄, filtered, and evaporated under reducedpressure. The crude material was purified by flash column chromatography(50% EtOAc in hexanes) to afford 2.98 g product in 70% yield.

Into the solution of S1 (2.13 g, 11.8 mmol) in 50 mL DMF was added S3(2.78 g, 13.0 mmol) and Cs₂CO₃ (11.54 g, 35.4 mmol). The mixture washeated at 80° C. overnight. Then the reaction was cooled to roomtemperature, and diluted with EtOAc. The solution was washed withsaturated NH₄Cl (50 mL×2) Aqueous phase was extracted with more EtOAc.Combined organic phase was washed with brine, dried over MgSO₄, followedby filtration and evaporation under reduced pressure. The crude materialwas purified by flash column chromatography (40% to 100% EtOAc inhexanes) to afford 3.94 g product in 85% yield.

S4 was taken up in trifluoroacetic acid (TFA) as 1M solution, which wasstirred at room temperature for 2 hours. The solution was concentratedunder reduced pressure, further on high-vac overnight. S6 was directlyused as starting material for the following synthesis without furtherpurification.

Amide Formation by HATU-Catalyzed Coupling Reaction

S6 was taken up in DMF as 1M solution, and 3 equivalence of Et₃N wasadded. The carboxylic acid (1.2 eq) was pre-mixed with HATU (2 eq) andEt₃N (5 eq) in DMF at the same concentration, which was stirred at roomtemperature for 10 min. Two solutions were then mixed together andfurther stirred at room temperature for 5 hours. The reaction wasdirectly subjected to prep. HPLC purification. The isolated product wasthen further purified by normal phase flash chromatography to affordproduct with desired purity for following biological tests.

Amide Formation by Acylation Using Acid Chloride

S6 (0.04 g, 0.1 mmol) was taken up in 2 mL dichloromethane. Et₃N (0.07mL, 0.5 mmol) was added, followed by addition of acetyl chloride (0.015mL, 0.2 mL). The reaction was kept at 0° C. stirring for 2 hours. Thenthe reaction was quenched by adding drops of water, followed byimmediate purification by flash column chromatography. The isolatedproduct 6 was further purified by HPLC to afford 19 mg product in 57%yield.

Reduction of Aromatic Nitro Group

S7 (0.49 g, 1.08 mmol) was dissolved in 10 mL AcOH/EtOH (1:1). Ironpowder (0.25 g, 4.39 mmol) was added in one portion. The reaction wasthen stirred at 50° C. for 1 hour. The iron powder was removed byfiltration. Filtrate was concentrated under reduced pressure. The crudematerial was then purified by normal phase flash column chromatography(10% to 40% MeOH in EtOAc), followed by reverse phase HPLC to afford0.22 g product S8 in 53% yield

Installation of Solubilizing Groups

S8 (0.11 g, 0.25 mmol) was dissolved in 5 mL dichloromethane. Et₃N(0.035 mL, 0.25 mmol) was added at −20° C.3-bromopropionyl chloride(0.03 mL, 0.25 mmol) in 1 mL DCM was added dropwisely. The reaction wasstirred at 0° C. for 3 h. Then it was quenched by addition of drops ofwater, then concentrated under reduced pressure. The crude product wasused for the next step without further purification.

Crude material from last step (0.06 g, 0.1 mmol) was dissolved in 1 mLDMF. Into the solution was added N-methylpiperazine (0.016 mL, 0.12mmol) and Et₃N (0.028 mL, 0.2 mmol). The reaction was stirred at 80° C.for 3 hours. The solution was directly subjected to reverse phase HPLCpurification, followed by normal phase flash column chromatography (20%to 60% MeOH in EtOAc with 0.5% Et₃N) to afford 0.043 g product 10 in 75%yield.

Using these procedures and variations thereof, the following compoundswere synthesized.

((R)-7-Chloro-3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one:white solid, 50% yield)¹H NMR (500 MHz, DMSO) δ 8.27 (d, J=12.8 Hz, 1H),8.16 (d, J=8.6 Hz, 1H), 7.75 (d, J=1.7 Hz, 1H), 7.58 (dd, J=8.6, 2.0 Hz,1H), 7.26 (dd, J=13.8, 6.7 Hz, 4H), 7.20-7.08 (m, 1H), 4.93 (d, J=5.9Hz, 1H), 4.04 (d, J=13.6 Hz, 2H), 3.92 (q, J=13.9 Hz, 1H), 3.65 (t,J=12.5 Hz, 1H), 3.28-3.06 (m, 2H), 2.86 (ddd, J=13.9, 8.6, 3.2 Hz, 1H),2.68-2.50 (m, 2H), 1.62-1.26 (m, 3H), 1.26-1.12 (m, 3H). ¹³C NMR (126MHz, DMSO) δ 168.92, 159.90, 150.16, 148.76, 146.43, 146.31, 138.70,128.22, 127.99, 127.96, 126.98, 126.68, 126.64, 125.99, 125.74, 125.69,120.12, 69.05, 69.00, 53.58, 40.82, 40.71, 39.99, 36.68, 36.01, 35.78,34.78, 34.65, 34.09, 33.95, 21.83, 21.63. LCMS (ESI) m/z 440.29 [(M+H)+;calcd for C₂₄H₂₇ClN₃O₃ ⁺: 440.17].

(7-Chloro-3-((1-(3-phenylpropanoyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one:white solid, 33% yield)¹H NMR (500 MHz, CDCl₃) δ 8.21 (d, J=8.6 Hz, 1H),7.93 (s, 1H), 7.70 (d, J=1.9 Hz, 1H), 7.46 (dd, J=8.6, 2.0 Hz, 1H),7.31-7.25 (m, 2H), 7.22-7.18 (m, 3H), 4.69 (d, J=13.4 Hz, 1H), 3.88-3.73(m, 3H), 3.00-2.92 (m, 2H), 2.91-2.82 (m, 1H), 2.60 (dp, J=14.3, 7.3 Hz,2H), 2.48 (td, J=13.1, 2.4 Hz, 1H), 2.17-2.04 (m, 1H), 1.70 (d, J=12.9Hz, 1H), 1.62 (d, J=12.8 Hz, 1H), 1.17 (qd, J=12.5, 4.3 Hz, 1H), 0.97(qd, J=12.5, 4.2 Hz, 1H). ¹³C NMR (126 MHz, DMSO) δ 169.56, 159.76,149.58, 148.95, 141.43, 138.88, 128.37, 128.18, 127.28, 126.30, 125.79,120.37, 50.79, 44.55, 40.74, 38.22, 35.01, 33.98, 30.87, 29.54, 28.84.LCMS (ESI) m/z 410.29 [(M+H)+; calcd for C₂₃H2₅CN₃O₂ ⁺: 410.16].

4-((7-Chloro-4-oxoquinazolin-3(4H)-yl)methyl)-1-(3-phenylpropanoyl)piperidine-4-carbonitrile:white solid, commercial compound)¹H NMR (500 MHz, DMSO) δ 8.44 (s, 1H),8.17 (d, J=8.6 Hz, 1H), 7.79 (d, J=2.0 Hz, 1H), 7.62 (dd, J=8.6, 2.1 Hz,1H), 7.25 (ddd, J=13.3, 7.9, 4.0 Hz, 4H), 7.20-7.12 (m, 1H), 4.46 (d,J=13.8 Hz, 1H), 4.36-4.24 (m, 2H), 3.97 (d, J=14.3 Hz, 1H), 3.05 (t,J=12.5 Hz, 1H), 2.80 (t, J=7.7 Hz, 2H), 2.73-2.57 (m, 3H), 1.88 (t,J=14.6 Hz, 2H), 1.61 (dtd, J=17.1, 13.0, 3.9 Hz, 2H). ¹³C NMR (126 MHz,DMSO) δ 170.41, 160.54, 149.90, 149.20, 141.81, 139.78, 128.99, 128.89,128.70, 128.13, 126.94, 126.34, 121.06, 120.79, 50.64, 42.38, 40.40,38.51, 34.30, 32.87, 32.28, 31.22. LCMS (ESI) m/z 435.29 [(M+H)+; calcdfor C₂₄H2₄ClN₄O₂ ⁺: 435.16].

(7-Chloro-3-((4-hydroxy-1-(3-phenylpropyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one:white solid, 13% yield)¹H NMR (500 MHz, MeOD) δ 8.29 (s, 1H), 8.21 (d,J=8.6 Hz, 1H), 7.69 (d, J=1.9 Hz, 1H), 7.54 (dd, J=8.6, 2.0 Hz, 1H),7.26 (q, J=7.1 Hz, 2H), 7.21-7.13 (m, 3H), 4.11 (s, 2H), 2.97 (d, J=12.0Hz, 2H), 2.76-2.59 (m, 6H), 1.98-1.81 (m, 4H), 1.63 (d, J=13.4 Hz, 2H).¹³C NMR (126 MHz, MeOD) δ 161.13, 149.97, 148.77, 140.38, 140.25,128.24, 128.13, 128.00, 127.52, 126.00, 120.22, 67.60, 56.16, 53.81,48.21, 32.27, 32.00, 25.90. LCMS (ESI) m/z 412.39 [(M+H)+; calcd forC₂₃H₂₇ClN₃O₂ ⁺: 412.18].

(7-chloro-3-((3-hydroxy-1-(3-phenylpropanoyl)pyrrolidin-3-yl)methyl)quinazolin-4(3H)-one:white solid, 8% yield)¹H NMR (500 MHz, DMSO) δ 8.29 (s, 1H), 8.17 (dd,J=8.6, 4.3 Hz, 1H), 7.76 (t, J=2.3 Hz, 1H), 7.59 (ddd, J=8.7, 7.5, 2.1Hz, 1H), 7.29-7.19 (m, 4H), 7.16 (t, J=6.8 Hz, 1H), 5.27 (s, 1H), 4.17(s, 1H), 4.14 (d, J=4.0 Hz, 1H), 3.51 (s, 2H), 3.32 (dt, J=23.6, 11.7Hz, 3H), 2.84-2.74 (m, 2H), 2.59-2.51 (m, 2H), 2.47-2.40 (m, 1H), 1.95(ddt, J=39.5, 12.7, 9.3 Hz, 1H), 1.76 (ddd, J=12.9, 11.0, 5.7 Hz, 1H).¹³C NMR (126 MHz, DMSO) δ 169.93, 169.76, 160.22, 160.13, 150.33,149.06, 149.04, 141.55, 141.51, 138.95, 128.45, 128.40, 128.35, 128.22,127.24, 126.27, 125.81, 120.46, 78.33, 76.84, 55.97, 55.57, 51.10,50.87, 44.46, 43.77, 36.06, 35.63, 35.06, 34.54, 30.29, 30.23. LCMS(ESI) m/z 412.29 [(M+H)+; calcd for C₂₂H₂₃ClN₃O₃ ⁺: 412.14].

(3-((1-Acetyl-4-hydroxypiperidin-4-yl)methyl)-7-chloroquinazolin-4(3H)-one:white solid, 57% yield)¹H NMR (500 MHz, DMSO) δ 8.40 (s, 1H), 8.15 (d,J=8.6 Hz, 1H), 7.75 (d, J=2.0 Hz, 1H), 7.57 (dd, J=8.6, 2.1 Hz, 1H),4.09-3.96 (m, 4H), 3.58 (d, J=13.4 Hz, 1H), 3.33-3.20 (m, 1H), 2.97-2.85(m, 1H), 1.98 (s, 3H), 1.56 (td, J=13.3, 4.3 Hz, 1H), 1.49-1.34 (m, 3H).¹³C NMR (126 MHz, DMSO) δ 167.66, 159.89, 150.25, 148.62, 138.65,128.19, 126.92, 125.88, 120.05, 68.98, 53.39, 41.40, 36.44, 34.61,33.93, 20.98. LCMS (ESI) m/z 336.18 [(M+H)+; calcd for C₁₆H₁₉ClN₃O₃ ⁺:336.11].

(7-Chloro-3-((4-hydroxy-1-(2-phenylacetyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one:white solid, commercial compound)¹H NMR (500 MHz, DMSO) δ 8.27 (s, 1H),8.15 (d, J=8.6 Hz, 1H), 7.75 (d, J=2.0 Hz, 1H), 7.57 (dd, J=8.6, 2.1 Hz,1H), 7.30-7.24 (m, 2H), 7.24-7.15 (m, 3H), 4.07-4.03 (m, 1H), 3.97 (dd,J=38.6, 13.8 Hz, 2H), 3.74-3.64 (m, 3H), 3.29-3.20 (m, 1H), 3.03-2.92(m, 1H), 1.51-1.27 (m, 4H). ¹³C NMR (126 MHz, DMSO) δ 168.31, 159.88,150.12, 148.71, 138.66, 135.76, 128.57, 128.19, 127.99, 126.93, 126.00,125.95, 120.07, 68.97, 53.45, 41.15, 39.35, 36.86, 34.61, 33.99. LCMS(ESI) m/z 412.29 [(M+H)+; calcd for C₂₂H₂₃ClN₃O₃ ⁺: 412.14].

(7-Chloro-3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one:white solid, commercial compound)¹H NMR (500 MHz, DMSO) δ 8.29 (s, 1H),8.18 (d, J=8.5 Hz, 1H), 7.71 (d, J=2.0 Hz, 1H), 7.56 (dd, J=8.6, 2.1 Hz,1H), 7.30-7.22 (m, 4H), 7.18-7.13 (m, 1H), 4.12-3.55 (m, 4H), 3.41-2.85(m, 3H), 2.62 (dd, J=14.9, 6.6 Hz, 1H), 2.54 (dd, J=14.9, 7.6 Hz, 1H),1.40 (t, J=15.9 Hz, 4H), 1.25 (d, J=7.0 Hz, 3H). ¹³C NMR (126 MHz, DMSO)δ 168.89, 159.94, 159.88, 150.11, 148.74, 146.42, 146.30, 138.68,128.20, 127.97, 127.94, 126.94, 126.66, 126.63, 125.98, 125.72, 125.67,120.10, 69.04, 68.99, 53.57, 40.82, 40.71, 39.99, 36.67, 35.99, 35.77,34.78, 34.64, 34.09, 33.95, 21.81, 21.61. LCMS (ESI) m/z 440.39 [(M+H)+;calcd for C₂₄H₂₇ClN₃O₃ ⁺: 440.17].

((S)-7-Chloro-3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one:white solid, 57% yield)¹H NMR (500 MHz, DMSO) δ 8.27 (d, J=12.8 Hz, 1H),8.16 (d, J=8.6 Hz, 1H), 7.75 (d, J=1.6 Hz, 1H), 7.58 (dd, J=8.6, 2.0 Hz,1H), 7.26 (dd, J=13.7, 6.6 Hz, 4H), 7.21-7.09 (m, 1H), 4.93 (d, J=5.8Hz, 1H), 3.99 (dd, J=43.9, 13.7 Hz, 2H), 3.91 (s, 1H), 3.65 (t, J=12.2Hz, 1H), 3.28-3.09 (m, 2H), 2.85 (d, J=13.2 Hz, 1H), 2.68-2.55 (m, 2H),1.60-1.25 (m, 3H), 1.20 (dd, J=6.9, 1.5 Hz, 3H). ¹³C NMR (126 MHz, DMSO)δ 168.89, 159.94, 159.88, 150.11, 148.74, 146.42, 146.30, 138.68,128.20, 127.97, 127.94, 126.94, 126.66, 126.63, 125.98, 125.72, 125.67,120.10, 69.04, 68.99, 53.57, 40.82, 40.71, 39.99, 36.67, 35.99, 35.77,34.78, 34.64, 34.09, 33.95, 21.81, 21.61. LCMS (ESI) m/z 440.29 [(M+H)⁺;calcd for C₂₄H₂₇ClN₃O₃ ⁺: 440.17].

((R)—N-(3-((4-Hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)propanamide:off-white solid, 75% yield)¹H NMR (500 MHz, DMSO) δ 10.57 (s, 1H), 8.20(d, J=13.1 Hz, 1H), 8.07 (d, J=8.7 Hz, 1H), 8.02 (d, J=1.6 Hz, 1H), 7.64(d, J=8.7 Hz, 1H), 7.25 (dd, J=12.4, 6.2 Hz, 4H), 7.13 (dd, J=18.7, 10.4Hz, 1H), 4.96 (d, J=5.8 Hz, 1H), 4.02 (d, J=13.6 Hz, 1H), 3.90 (q,J=14.0 Hz, 2H), 3.63 (dd, J=29.0, 16.2 Hz, 1H), 3.27-3.12 (m, 2H), 2.86(dd, J=17.7, 14.8 Hz, 1H), 2.69-2.61 (m, 2H), 2.61-2.52 (m, 3H),2.48-2.22 (m, 8H), 2.16 (s, 3H), 1.58-1.26 (m, 4H), 1.20 (d, J=6.4 Hz,3H). ¹³C NMR (126 MHz, DMSO) δ 170.76, 168.83, 159.92, 159.87, 149.10,148.73, 146.41, 146.28, 144.06, 127.93, 127.90, 126.97, 126.62, 126.60,125.69, 125.63, 118.04, 116.26, 114.45, 69.03, 68.98, 54.37, 53.22,51.96, 45.30, 40.81, 40.71, 39.97, 36.67, 35.95, 35.74, 34.77, 34.64,34.09, 34.01, 33.95, 21.79, 21.61. LCMS (ESI) m/z 575.32 [(M+H)⁺; calcdfor C₃₂H₄₃N₆O₄ ⁺: 575.33].

((S)—N-(3-((4-Hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)propanamide:off-white solid, 26% yield)¹H NMR (500 MHz, DMSO) δ 10.52 (s, 1H), 8.19(d, J=13.1 Hz, 1H), 8.08 (d, J=8.7 Hz, 1H), 8.02 (d, J=2.0 Hz, 1H), 7.62(dd, J=8.8, 1.4 Hz, 1H), 7.25 (dd, J=12.6, 6.3 Hz, 4H), 7.19-7.08 (m,1H), 4.93 (s, 1H), 4.08-3.97 (m, 1H), 3.90 (q, J=14.0 Hz, 2H), 3.64 (t,J=12.9 Hz, 1H), 3.30-3.09 (m, 2H), 2.95-2.81 (m, 1H), 2.69-2.60 (m, 2H),2.61-2.52 (m, 3H), 2.47-2.25 (m, 8H), 2.15 (s, 3H), 1.58-1.26 (m, 4H),1.24-1.14 (m, 3H). ¹³C NMR (126 MHz, DMSO) δ 170.76, 168.82, 159.88,149.11, 148.74, 146.41, 146.29, 144.04, 127.94, 127.91, 127.00, 126.63,125.69, 125.64, 118.04, 116.28, 114.46, 68.99, 54.43, 53.24, 52.02,45.38, 40.82, 40.71, 39.96, 36.68, 35.96, 35.75, 34.77, 34.64, 34.03,21.80, 21.61. LCMS (ESI) m/z 575.32 [(M+H)⁺; calcd for C₃₂H₄₃N₆O₄ ⁺:575.33].

(N-(3-((4-Hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)propenamide:white solid, 16% yield)¹H NMR (500 MHz, DMSO) δ 10.84 (s, 1H), 8.25 (d,J=13.2 Hz, 1H), 8.15-7.99 (m, 2H), 7.69 (d, J=8.7 Hz, 1H), 7.28-7.25 (m,4H), 7.19-7.10 (m, 1H), 4.05-3.88 (m, 3H), 3.65 (t, J=12.8 Hz, 1H),3.28-3.14 (m, 10H), 2.92-2.85 (m, 3H), 2.79 (s, 3H), 2.64-2.53 (m, 4H),1.53-1.28 (m, 4H), 1.20 (d, J=6.0, 3H). ¹³C NMR (126 MHz, DMSO) δ169.60, 160.66, 160.61, 158.82, 158.57, 149.95, 149.34, 147.15, 147.02,144.63, 128.69, 128.66, 127.69, 127.38, 127.35, 126.44, 126.39, 118.90,117.14, 115.37, 69.76, 69.71, 53.94, 41.57, 41.46, 40.88, 40.70, 37.42,36.71, 36.49, 35.50, 35.37, 34.81, 34.66, 22.53, 22.34. LCMS (ESI) m/z575.32 [(M+H)⁺; calcd for C₃₂H₄₃N₆O₄ ⁺: 575.33].

(N-(3-((4-Hydroxy-1-(4-methyl-3-phenylpentanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)propenamide:off-white solid, 40% yield)¹H NMR (500 MHz, DMSO) δ 10.65 (s, 1H), 8.21(d, J=16.9 Hz, 1H), 8.11 (dd, J=8.7, 1.9 Hz, 1H), 8.05 (s, 1H), 7.65 (d,J=8.7 Hz, 1H), 7.23 (td, J=7.6, 2.9 Hz, 2H), 7.15 (t, J=6.5 Hz, 2H),7.13-7.06 (m, 1H), 4.04-3.93 (m, 2H), 3.85 (s, 1H), 3.75-3.61 (m, 1H),3.58-2.98 (m, 10H), 2.91-2.55 (m, 10H), 1.88-1.80 (m, 1H), 1.53-1.25 (m,3H), 1.12-1.06 (m, 1H), 0.89 (t, J=7.2 Hz, 3H), 0.70-0.61 (m, 3H). ¹³CNMR (126 MHz, DMSO) δ 169.94, 169.86, 160.61, 160.53, 149.95, 149.29,144.51, 144.09, 144.03, 128.82, 128.79, 128.24, 128.16, 127.81, 126.31,118.89, 117.21, 115.33, 69.76, 69.66, 54.04, 50.83, 49.24, 49.08, 48.75,42.48, 41.51, 37.43, 37.33, 36.01, 35.94, 35.55, 35.31, 34.72, 34.65,32.83, 32.63, 32.39, 21.24, 21.19, 20.76, 20.56. LCMS (ESI) m/z 603.43[(M+H)⁺; calcd for C₃₄H₄₇N₆O₄ ⁺: 603.37].

((R)—N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-morpholinopropanamide:off-white solid, 65% yield)¹H NMR (500 MHz, DMSO) δ 10.52 (s, 1H), 8.20(d, J=13.3 Hz, 1H), 8.07 (d, J=8.7 Hz, 1H), 8.04 (d, J=1.8 Hz, 1H), 4.96(d, J=6.4 Hz, 1H), 4.02 (d, J=13.7 Hz, 1H), 3.90 (q, J=14.0 Hz, 2H),3.73-3.60 (m, 1H), 3.59-3.55 (m, 4H), 3.18 (ddd, J=21.0, 18.3, 9.2 Hz,2H), 2.93-2.80 (m, 1H), 2.65 (t, J=6.9 Hz, 2H), 2.61-2.54 (m, 3H), 2.41(m, 4H), 1.40 (dddd, J=41.2, 26.0, 16.8, 9.5 Hz, 4H), 1.19 (dd, J=6.9,1.7 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 170.62, 168.81, 160.00, 149.15,148.76, 146.44, 146.31, 144.09, 118.13, 116.29, 114.51, 69.06, 65.93,53.73, 52.79, 40.85, 40.75, 39.98, 36.71, 36.00, 35.78, 34.79, 34.66,34.11, 33.98, 33.81, 21.83, 21.64. LCMS (ESI) m/z 562.32 [(M+H)⁺; calcdfor C₃₁H₄₀N₅O₅: 562.30].

((R)-3-(Dimethylamino)-N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)propanamide:off-white solid, 36% yield)¹H NMR (500 MHz, DMSO) δ 10.47 (s, 1H), 8.18(dd, J=13.0, 7.3 Hz, 1H), 8.06 (d, J=8.7 Hz, 1H), 8.02 (d, J=2.0 Hz,1H), 7.63-7.59 (m, 1H), 7.24 (dd, J=12.6, 6.3 Hz, 4H), 7.17-7.09 (m,1H), 4.92 (s, 1H), 4.07-3.95 (m, 1H), 3.89 (q, J=14.0 Hz, 2H), 3.63 (t,J=13.0 Hz, 1H), 3.18 (ddd, J=20.8, 18.0, 9.1 Hz, 2H), 2.86 (ddd, J=13.8,10.5, 5.5 Hz, 1H), 2.60-2.54 (m, 3H), 2.54-2.47 (m, 2H), 2.17 (s, 6H),1.56-1.23 (m, 4H), 1.24-1.15 (m, 3H). ¹³C NMR (126 MHz, DMSO) δ 170.76,168.83, 159.89, 149.09, 148.74, 146.42, 146.29, 144.07, 127.94, 127.92,126.97, 126.61, 125.70, 125.64, 118.06, 116.26, 114.46, 68.99, 54.59,53.29, 44.63, 40.82, 40.72, 39.98, 39.96, 36.68, 35.97, 35.75, 34.78,34.65, 34.61, 34.11, 33.96, 21.80, 21.61. LCMS (ESI) m/z 520.31 [(M+H)⁺;calcd for C₂₉H₃₈N₅O₄ ⁺: 520.29].

(N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(1H-imidazol-1-yl)propenamide:white solid, 19% yield)¹H NMR (500 MHz, DMSO) δ 10.62 (s, 1H), 9.16 (s,1H), 8.22 (d, J=12.9 Hz, 1H), 8.09 (d, J=8.7 Hz, 1H), 8.00 (s, 1H), 7.80(s, 1H), 7.67 (s, 1H), 7.62 (d, J=8.8 Hz, 1H), 7.28-7.25 (m, 4H), 7.15(d, J=3.6 Hz, 1H), 4.03-3.87 (m, 3H), 3.65 (t, J=12.4 Hz, 1H), 3.26-3.14(m, 2H), 3.08 (t, J=6.3 Hz, 2H), 2.90-2.85 (m, 1H), 2.64-2.49 (m, 4H),1.54-1.25 (m, 4H), 1.20 (d, J=5.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ169.60, 169.43, 160.60, 160.55, 158.88, 158.60, 149.99, 149.25, 147.15,147.03, 144.33, 136.33, 128.68, 128.66, 127.85, 127.37, 127.35, 126.44,126.39, 122.58, 120.32, 118.86, 117.25, 115.33, 69.77, 69.71, 54.04,44.94, 41.55, 41.44, 40.71, 37.41, 36.70, 36.57, 36.49, 35.52, 35.39,34.80, 34.66, 22.53, 22.35. LCMS (ESI) m/z 543.22 [(M+H)⁺; calcd forC₃₀H₃₅N₆O₄ ⁺: 543.27].

((N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(piperidin-1-yl)propenamide:white solid, 18% yield)¹H NMR (500 MHz, DMSO) δ 10.69 (s, 1H), 9.32 (s,1H), 8.23 (d, J=12.8 Hz, 1H), 8.11 (dd, J=8.7, 1.9 Hz, 1H), 8.05 (s,1H), 7.65 (d, J=8.7 Hz, 1H), 7.19-7.31 (m, 4H), 7.15 (s, 1H), 4.03-3.89(m, 3H), 3.65 (t, J=12.4 Hz, 1H), 3.48-3.40 (m, 4H), 3.26-3.15 (m, 2H),2.98-2.89 (m, 5H), 2.64-2.51 (m, 2H), 1.84 (d, J=13.3 Hz, 2H), 1.68-1.62(m, 3H), 1.55-1.31 (m, 5H), 1.21 (d, J=6.6 Hz, 3H). ¹³C NMR (126 MHz,DMSO) δ 169.60, 169.22, 160.61, 160.56, 158.87, 158.59, 150.01, 149.29,147.15, 147.03, 144.41, 128.68, 128.66, 127.87, 127.37, 127.35, 126.44,126.39, 118.89, 117.27, 115.38, 69.77, 69.72, 54.05, 52.89, 52.05,41.55, 41.45, 40.69, 37.41, 36.71, 36.49, 35.53, 35.40, 34.81, 34.67,31.22, 23.00, 22.53, 22.35, 21.65. LCMS (ESI) m/z 560.22 [(M+H)⁺; calcdfor C₃₂H₄₂N₅O₄ ⁺: 560.32].

Example 3: Biological Assays USP7 Enzyme Expression and Purification

A construct of human USP7 covering residues 208-560 in the pET28aLICvector was overexpressed in E. coli BL21 (DE3) in terrific broth (TB)medium in the presence of 50 μg/ml of kanamycin. Cells were grown at 37°C. to an OD of 0.8, cooled to 17° C., induced with 500 μMisopropyl-1-thio-D-galactopyranoside (IPTG), incubated overnight at 17°C., collected by centrifugation, and stored at −80° C. Cell pellets weresonicated in buffer A (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 10mM Imidazole, and 3 mM BME) and the resulting lysate was centrifuged at30,000×g for 40 min. Ni-NTA beads (Qiagen) were mixed with lysatesupernatant for 30 min and washed with buffer A. Beads were transferredto an FPLC-compatible column and the bound protein was washed with 15%buffer B (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 300 mMImidazole, and 3 mM BME) and eluted with 100% buffer B. Thrombin wasadded to the eluted protein and incubated at 4° C. overnight. The samplewas then concentrated and passed through a Superdex 200 16/60 column (GEHealthcare) in a buffer containing 20 mM HEPES pH 7.5, 200 mM NaCl, 5%glycerol, and 1 mM TCEP. Fractions were pooled, concentrated and frozenat −80° C.

USP7 full length (aa 0001.1102) in pET28aLIC was transformed inBL21(DE3) cells. An overnight culture was used to inoculate one liter ofTB supplemented with 50 μg/ml kanamycin. Cells were grown at 37° C. tillthey reached optical density (OD) 0.6 at 600 nm. Protein expression wasinitiated by the addition of 0.4 mM IPTG. Cells were then grown for16-20 hours at 17° C. prior collection by centrifugation. Cell pelletswere washed in PBS and resuspended in 25 mM HEPES pH 7.5, 500 mM NaCl,10% glycerol and 1 mM TCEP, 10 mM Imidazole, 0.1% IGEPAL sonicated andincubated with Ni-Nta beads (Quiagen) for 30 min at 4° C. Beads werewashed with 10% buffer B (25 mM HEPES pH 7.5, 500 mM NaCl, 10% glyceroland 1 mM TCEP, 250 mM Imidazole) and eluted with 100% buffer B. Proteincontaining fractions were concentrated and loaded on Superdex 200 10/300GL column in a buffer containing 20 mM HEPES pH 7.5, 200 mM NaCl, 5%glycerol, and 1 mM TCEP. Fractions were pooled, concentrated and frozenat −80° C.

Site Directed Mutagenesis

Amino acid mutations of catalytic domain and full length USP7 wereintroduced by PCR using QuikChange site-directed mutagenesis kit(Stratagene, La Jolla, Calif.) following manufacturer's protocol. Table1 reports primer used for each mutation created.

TABLE 1 SEQ ID Orientation Mutation Primer NO: of Primers Q351SGAAGATTATTATGATATCTCGCTAAGTATCAAAGG 1 forwardCCTTTGATACTTAGCGAGATATCATAATAATCTTC 2 reverse M407KCCAGTGTTACATCTACAACTGAAGAGATTTATGTATGACCC 3 forwardGGGTCATACATAAATCTCTTCAGTTGTAGATGTAACACTGG 4 reverse M410SCTACAACTGATGAGATTTAGTTATGACCCTCAGACGGACC 5 forwardGGTCCGTCTGAGGGTCATAACTAAATCTCATCAGTTGTAG 6 reverse M407K/M410SCCAGTGTTACATCTACAACTGAAGAGATTTAGTTATGACCCTCAGACGGACC 7 forwardGGTCCGTCTGAGGGTCATAACTAAATCTCTTCAGTTGTAGATGTAACACTGG 8 reverse K420ACCCTCAGACGGACCAAAATATCGCGATCAATGATAGGTTTGAATTCC 9 forwardGGAATTCAAACCTATCATTGATCGCGATATTTTGGTCCGTCTGAGGG 10 reverse H456ACTTCATGCAGTCCTGGTTGCTAGTGGAGATAATCATGGTGG 11 forwardCCACCATGATTATCTCCACTAGCAACCAGGACTGCATGAAG 12 reverse H461ACTGGTTCATAGTGGAGATAATGCTGGTGGACATTATGTGG 13 forwardCCACATAATGTCCACCAGCATTATCTCCACTATGAACCAG 14 reverse Y514ACGACACTGCACTAATGCTGCCATGTTAGTCTACATCAGGG 15 forwardCCCTGATGTAGACTAACATGGCAGCATTAGTGCAGTGTCG 16 reverse

Isothermal Titration Calorimetry

The binding affinity of protein/ligand was measured by adding 0.02 mMprotein in cell and titrating with 0.2 mM ligand in the syringe using anAuto-ITC200 microcalorimeter (Malvern) at 20° C. (FIG. 2C). Proteins andligands were prepared within ITC buffer containing 20 mM HEPES pH 7.5,150 mM NaCl, and 2% DMSO. The data were fit using Origin 7.0 software.ITC results are summarized in Table 2.

TABLE 2 DS (cal/ DH n mol• Ligand Kd (μM) (kcal/mol) (stoichiometry) K)10 0.104 ± 0.015 −15.1 ± 0.1 1.06 ± 0.01 119.6 A 7.614 ± 3.216 −5.4 ±1.3 0.98 ± 0.15 5.3 7 No binding detected 8 1.838 ± 0.895 −3.9 ± 0.22.46 ± 0.09 13.1 9 0.797 ± −0.097 −13.5 ± 0.3 0.96 ± 0.01 −17.3

Selectivity Profiling

Selectivity profiling (DUBProfiler) was performed by Ubiquigent usingthe manufacturer's protocols. FIG. 2B illustrates the dose-dependentinhibition of the USP7 catalytic domain by compounds A, 10 and 11. FIG.2D illustrates inhibitory activity of compound 10 across a panel of 41purified DUBs using ubiquitin-rhodamine (Ub-Rho) as substrate.

Competitive Activity Based Protein Profiling

HEK 293T cells were pelleted, washed with PBS, lysed on ice (50 mM TrispH 7.6, 150 mM NaCl, 5 mM MgCl₂, 0.5 mM EDTA, 0.5% NP-40, 10% glycerol,1 mM TCEP, phosphatase inhibitor cocktails (Sigma P5726 and Calbiochem524624), and protease inhibitors (pepstatin, leupeptin, PMSF, andaprotinin), and clarified by centrifugation. Protein content wasquantified by BCA, and 50 ug of lysate was diluted into 30 uL labelingbuffer (50 mM Tris pH 7.6, 5 mM MgCl₂, 0.5 mM EDTA, 250 mM sucrose, 1 mMTCEP) and incubated at room temperature with shaking with the indicatedinhibitors for 30 minutes. Samples were then supplemented with 1 uMHA-Ub-VS and incubated at room temperature with shaking for 15 minutes.Reactions were quenched with 4×LDS sample buffer (Thermo Fisher B0007)supplemented with 10% BME, vortexed vigorously, and heated to 95° C. for5 minutes. Samples were resolved by SDS-PAGE and analyzed by Westernblot with the indicated antibodies (FIG. 5C).

Cell Treatments

MCF7 and MM.1S were grown in RPMI supplemented with 10% Fetal Bovineserum (FBS) and antibiotics. Cells were treated with DMSO or differentconcentrations of compounds 10 and 11 for 6 (MM1S) or 16 (MCF7) hours inpresence or absence of cycloheximide. For the experiments in whichcycloheximide was used, cells were treated with compounds for 4 (MM1S)or 14 (MCF7) hours prior to the addition of 50 ug/ml of cycloheximide.At 6 or 16 h time point cells were washed in PBS and lysed in modifiedRIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 20 mMTris, 150 mM NaCl, 1 mM EDTA) containing phosphatase inhibitor cocktails1 and 2 (Sigma), and protease inhibitors. Protein concentrations werequantified using the BCA protein assay kit (Pierce) and samples wereprobed by immunoblotting using mdm2 (santa cruz sc-965), p53 (cellsignaling 9282), p21 (cell signaling 2947), GAPDH (cell signaling 2118),USP7 (cell signaling 4833) antibodies (FIGS. 6A-D).

Peripheral Blood Mononuclear Cell Testing

Peripheral blood mononuclear cells (PBMCs) were generously provided byDr. Steven Treon and Dr. Guang Yang. PBMCs from normal individuals wereisolated by density gradient centrifugation through Ficoll-Plaque Plus(Amersham Pharmacia Biotech AB, Uppsala, Sweden) at 400×g for 25minutes, followed by two washes in PBS. Cells were then maintained inRPMI+10% FBS, supplemented with 10% FBS. Primary cells were obtainedthrough written consent under approval of the Dana-Farber CancerInstitute Institutional Review Board. The trypan blue exclusion assayhas been previously described (Weisberg et al., 2002) and was used forquantification of PBMCs prior to seeding for CellTiter-Glo LuminescentCell Viability assays (Promega, Madison, Wis.). These assays were usedfor proliferation studies and carried out according to manufacturerinstructions. Cell viability is reported as percentage of control(untreated) cells, and error bars represent the standard deviation foreach data point.

Ub-AMC Assay

USP7 and mutants were tested for their activity in Ubiquitin-AMC assayin presence or absence of inhibitors. For this assay USP7 catalyticdomain WT or mutant was used at the following concentrations: 250 nMUSP7 WT, M407K, M407K/M410S or Q351S, 125 nM H461A, 600 nM Y514A and 10nM M410S. For the same assay USP7 full length WT and Q351 mutant wereused at 50 nM. USP7 variants were pre-incubated with differentconcentrations of inhibitors or DMSO as a control in 50 mM HEPES pH7.6,0.5 mM EDTA, 11 uM ovalbumin, 5 mM DTT. The reaction was incubated 30min at room temperature prior to the addition of 2 uM Ubiquitin-AMC(Boston Biochem) substrate. The initial rate of the reaction wasmeasured by collecting fluorescence data at one minute interval over30-minute period using a Clariostar fluorescence plate reader atexcitation and emission wavelength of 345 and 445 nm respectively. Thecalculated initial rate values were plotted against inhibitorconcentrations to determine IC₅₀s. All the experimental data wereplotted using Prism GraphPad. FIG. 2A illustrates the structure guidedoptimization for the inhibition of USP7. Compound A (WO 2013/030218)shown below was optimized to arrive at compounds 10 and 11.

FIGS. 3A-C illustrate the binding of compound 10 to USP7. FIGS. 4A-Dillustrate the binding and dose response inhibition of compounds A and10. FIGS. 5A-C illustrate the microsome stability and the USP7 bindingability of compound A and the presently disclosed compounds.

Table 3: USP7 activity of exemplary compounds in USP7 assay. ++++indicates an IC₅₀ of less than about 0.2 μM, +++ indicates an IC₅₀ fromabout 0.2 μM to about 1 μM, ++ indicates an IC₅₀ from about 1 μM toabout 10 μM, and + indicates an IC₅₀ greater than 10 μM. ND refers tonot disclosed.

TABLE 3 Compound IC₅₀ (μM)

1 ++++

2 ++++

3 ND

4 +

5 +

6 +

7 +

8 ++++

9 ++++

10 ++++

11 ++++

12 +++

13 +++

14 ++++

15 ++++

16 ++++

17 ++++

18 ++

19 ++

20 +

21 +

22 +

23 +

24 +

25 +

26 +

27 +

28 +

29 +++

30 ++

31 +

32 +

33 +

34 +

35 +

36 +

37 +

38 +++

39 +++

40 +

41 +

42 +

43 ND

44 +

45 ND

46 +

47 ++++

48 ++++

49 +

50 ++

51 +

52 +

53 +

54 +++

55 +

56 +

57 +

58 +

59 +

60 +

61 +

62 +

63 +

64 ++

65 +

66 +

67 +

68 +

69 +

70 +

71 +

72 +++

73 +

74 +

75 +

76 +

77 +

78 +

79 +

80 +

81 +

82 ++

83 +

84 ++

85 +

86 +++

87 +

88 +

89 +

90 +

91 +++

92 ++

93 ++

94 ++

95 +

96 +

97 +

98 +++

99 ++

100 +

101 +++

102 +++

103 ++

104 ++

105 +

Further exemplary compounds include, but are not limited to, those givenin Table 4 below.

TABLE 4 Compound

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

1. A compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: R₁ is H,halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NO₂, —NH₂, CN,—NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈, wherein each alkyl isindependently optionally substituted with one or more R₉; R₂ is H,halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NH₂, —NO₂, CN,—NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈, wherein each alkyl isindependently optionally substituted with one or more R₉; R₃ is H,halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NH₂, —NO₂, CN,—NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈; wherein each alkyl isindependently optionally substituted with one or more R₉; R₄ is H,halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, —OH, —NO₂, —NH₂, CN,—NR₇C(═O)alkyl, —C(═O)NR₇alkyl, or —NR₇R₈; wherein each alkyl isindependently optionally substituted with one or more R₉; wherein R₁,R₂, R₃ and R₄ are not simultaneously H; R₅ is H, halogen, —CN, —OR₇, or—NR₇R₈; R₆ is alkyl, —C(═O)R₁₀, —C(═S)R₁₀, —C(O)NR₇R₈, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl; wherein the cycloalkyl,heterocycloalkyl, aryl, heteroaryl are each independently optionallysubstituted with one or more R₁₁; and wherein the alkyl is substitutedwith one or more R₁₂; each R₇ and R₈ is independently H, acyl,alkylacyl, alkenyl, alkenylacyl, or alkyl; each R₉ is independently ateach occurrence azido, —NR₇R₈, alkoxy, —(OCH₂CH₂)_(m)alkyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl and alkoxy areeach independently optionally substituted with one or more substituentsselected from alkoxy, haloalkoxy, halogen, and —OH; and wherein thecycloalkyl, heterocycloalkyl, aryl and heteroaryl are each independentlyoptionally substituted with one or more substituents selected fromalkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —N₃, and —OH; R₁₀ isalkyl, alkenyl, alkynyl, —NR₇R₈, cycloalkyl, heterocycloalkyl, aryl,amino, heteroalkyl, alkylamino, aminoalkyl or heteroaryl, wherein thealkyl, alkenyl, and alkynyl are each independently optionallysubstituted with one or more R₁₃; and wherein the cycloalkyl,heterocycloalkyl, aryl and heteroaryl are each independently optionallysubstituted with one or more R₁₂; each R₁₁ is independently at eachoccurrence alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —NO₂, or —OH;each R₁₂ is independently at each occurrence aryl or heteroaryl, whereinthe aryl and heteroaryl are each independently optionally substitutedwith one or more substituents selected from alkyl, haloalkyl, alkoxy,haloalkoxy, halogen, and —OH; each R₁₃ is independently at eachoccurrence —OH, alkoxy, heteroalkyl, aryloxy, —NH₂, arylalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —O-heteroaryl, —NR₇aryl,—NR₇heteroaryl, or —NR₇C(═O)R₁₄, wherein the cycloalkyl,heterocycloalkyl, aryl, heteroalkyl, and heteroaryl are eachindependently optionally substituted with one or more substituentsselected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —NO₂, and—OH; R₁₄ is alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, cycloalkyl,aryl, heterocyclyl, or heteroaryl, wherein the aryl and heteroaryl areeach independently optionally substituted with one or more R₁₅; andwherein the alkyl, alkenyl, and alkynyl are each independentlyoptionally substituted with one or more substituents selected fromhalogen and —OH; each R₁₅ is independently at each occurrence halogen,alkyl, CN, —C(═O)alkyl, or —C(═O)alkenyl, wherein the alkyl and alkenylis each independently substituted with one or more substituents selectedfrom halogen and —OH; m is 1, 2, or 3; and n is 0 or 1; provided that:(i) if R₂ is —NO₂, —NHC(O)Me or —NH₂, and R₁, R₂, and R₄ are each H; orR₁ is Me and R₂, R₃, and R₄ are each H; then R₆ is not —C(O)R₁₀ whereR₁₀ is —(CH₂)—(CHMe)-phenyl; (ii) when R₂ is Cl, R₁, R₃ and R₄ are eachH, R₆ is —C(═O)R₁₀, and R₁₀ is (C₂-C₃)alkyl substituted with one R₁₃;then R₁₃ is not unsubstituted cyclopentyl, unsubstituted phenyl orunsubstituted 2-thiophenyl; and (iii) when R₂ is Cl, and R₁, R₃ and R₄are each H; then R₆ is —C(═O)R₁₀, R₁₀ is not 1-ethylpropyl.
 2. Thecompound of claim 1, wherein R₁ is H, —NR₇C(═O)alkyl, or —NR₇R₈. 3.(canceled)
 4. The compound of claim 1, wherein R₃ is H, —NO₂, or —NR₇R₈.5. (canceled)
 6. The compound of claim 1, wherein R₄ is H.
 7. Thecompound of claim 1, wherein each R₉ is independently at each occurrence—NR₇R₈, alkoxy, —(OCH₂CH₂)_(m)alkyl, heterocycloalkyl, or heteroaryl,wherein the heterocycloalkyl or heteroaryl are each independentlyoptionally substituted with one or more substituents selected fromalkyl, alkoxy, and —N₃.
 8. (canceled)
 9. (canceled)
 10. The compound ofclaim 1, wherein the compound has a structure of Formula (Ia):

or a pharmaceutically acceptable salt thereof.
 11. The compound of claim1, wherein R₂ is selected from halogen, —NH₂, —NO₂, CN, —NR₇C(═O)alkyland —C(═O)NR₇alkyl, wherein each alkyl is independently optionallysubstituted with one or more R₉. 12-15. (canceled)
 16. The compound ofclaim 1, wherein R₂ is —NR₇C(═O)alkyl or —C(═O)NR₇alkyl: the alkyl issubstituted with one R₉: R₉ is azido, —NR₇R₈, heterocycloalkyl orheteroaryl; and R₈ is H. 17-20. (canceled)
 21. The compound of claim 1,wherein R₂ is —NR₇C(═O)alkyl or —C(═O)NR₇alkyl: the alkyl is substitutedwith one R₉; and R₇ is acyl, alkylacyl, or alkenylacyl.
 22. (canceled)23. The compound of claim 1, wherein the compound has a structure ofFormula (Ib):

or a pharmaceutically acceptable salt thereof.
 24. The compound of claim1, wherein R₅ is H, CN, —OH, or —NR₇R₈.
 25. (canceled)
 26. (canceled)27. The compound of claim 1, wherein R₇ and R₈ are each H or alkyl. 28.The compound of claim 1, wherein R₆ is alkyl, —C(═O)R₁₀, —C(═S)R₁₀,aryl, or heteroaryl.
 29. (canceled)
 30. The compound of claim 28,wherein R₁₀ is alkyl, alkenyl, amino, alkylamino, alkynyl, cycloalkyl,heteroalkyl, heteroaryl, or aminoalkyl, each optionally substituted withone or more R₁₃. 31-36. (canceled)
 37. The compound of claim 1, whereinR₁₀ is alkyl, alkenyl, alkynyl, —NR₇R₈, cycloalkyl, or heterocycloalkyl,each optionally substituted with one or more R₁₃; and each R₁₃ isindependently at each occurrence —OH, alkoxy, aryloxy, —NH₂, arylalkyl,cycloalkyl, aryl, heteroaryl, or —NR₇C(═O)R₁₄.
 38. The compound of claim37, wherein each R₁₄ is independently at each occurrence alkyl,haloalkyl, arylalkyl, alkenyl, heterocyclyl, or heteroaryl.
 39. Thecompound of claim 10, wherein: R₂ is Cl, —NO₂, —NH₂ or —NR₇C(═O)alkyl,wherein the alkyl is optionally substituted with one or more R₉; R₆ is—C(═O)R₁₀; and R₁₀ is alkyl, alkenyl, alkynyl, cycloalkyl, andheterocycloalkyl, wherein the alkyl, alkenyl, and alkynyl are eachoptionally substituted with one or more R₁₃; and wherein the cycloalkyland heterocycloalkyl are each optionally substituted with one or moreR₁₂.
 40. (canceled)
 41. A compound selected from: Compound

or a pharmaceutically acceptable salt thereof.
 42. (canceled)
 43. Apharmaceutical composition comprising a compound of claim 1, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 44. A method of treating a disease or disordermodulated by USP7 comprising, administering to a subject in need thereofa compound of claim 1 or a pharmaceutically acceptable salt thereof. 45.(canceled)
 46. (canceled)
 47. A method of treating cancer, comprisingadministering to a subject in need thereof a compound of claim 1 or apharmaceutically acceptable salt thereof. 48-52. (canceled)